The Zulauf Ag located in Schinznach Dorf is one of the largest tree nurseries in Switzerland. The company, existing since 1879, reached a size of 30 ha and contains nowadays more than half a million plants (Zulauf AG, 2018). The tree nursery has a cycle within their irrigation system. The irrigation water which isn’t adsorb by the plants infiltrates directly or after leaking from the plant pots into the ground in whom the water is collected by an impermeable interlayer and guided to a pond. From this pond the water will then again be gathered for the irrigation only running through broad filter. External water is only needed to compensate the loss through transpiration and is drawn from the groundwater as Johannes Zulauf has told.
Within the irrigation system are several spots which are interesting for a research related to the microorganism community. One of those is the pond. As Mr. Zulauf told the pond use to be 30m deep but as recently discovered it has been filled by sediments up to 6m within a relatively brief period of time. Microorganism are playing a significant role in the decomposition of organic matter and are affected by the presence of pesticides (A. Widenfalk, 2005). Is it possible that pesticides accumulated inside the irrigation system altered the microorganism community and led to a reduced microbial activity and therefore to a fast filling of the pond? Therefor the microorganism community has to be characterized.
Literature describes diverse ways to characterize a bacteria community, first it’s crucial if free-living bacteria or a biofilm is investigated. A biofilm is a thin layer coated to a surface containing viable and nonviable microorganism trapped in a matrix of organic matter for example exopolymers (Biofilm, 2012).
Sample drawing
Samples for microbiological analysis must always be collected in sterile glass or polypropylene bottles. After the samples were taken they must be preserved at a temperature of approximately 4 °C till they attain in the laboratory. Microorganism are affected by their environment, therefore parameters such as pH and temperature has also to be measured. (Centre d'expertise en analyse environnmentale du Québece, 2009)
General appearance
If the microorganism formed a biofilm its useful to have a look at the general appearance. Through staining the biofilm with INT, active cells can be made visible. INT is reduced to INT-formazan of an orange colour by the active electron transport. The spatial distribution of active cells can then be determined with light microscopy. This method distinguishes itself with simplicity, rapidity and the possibility to observe the biomass without preliminary treatment but has a disadvantage in a low resolution. Other methods just as scanning electron microscopy (SEM) deliver more precise data but also requires a slow and complex sample preparation (V. Lazarova et al., 1995) which includes drying and therefor deform the hydrated structure (K. Ratnayake et al. 2012).
Total biomass
Another important parameter is the total biomass it can be measured by the thicknes of a biofilm. Phyiscal methods are are light microscopy, which is a fast method but only works on thin homogenous layers, electrical conductance, which gives more precise information but only works on metallic surfaces, or or the thermal resistance. A further physical method is to weigh the dry weight, always considering that inert mass and adsorbed organic matter is also measured. Combining the data of the biofilm thickness and the dry weight, the density can be estimated. (V. Lazarova et al., 1995)
A method for measuring free-living and biofilm biomass is the standard plate count of colony forming unity (CFU). This is one of the best known microbiological techniques and is used routinely for the analysis of bacteria population. (V. Lazarova et al., 1995) To be able to count the microorganism, the sample gets elapsed on a petri dish. The single bacteria are spreaded on the medium and still growing. After an incubation time of about 18 -28 hours the single bacteria formed a colony which now can be counted (S. Sieuwerts et al., 2008) CFU has the disadvantage that only cells capable reproducing themselves are measured, which leads to an underestimation. Beside the CFU methode also spectrophotometry and turbidimetry can be used for measuring free-living biomass. (V. Lazarova et al., 1995)
Literature describes diverse ways to characterize a bacteria community, first it’s crucial if free-living bacteria or a biofilm is investigated. A biofilm is a thin layer coated to a surface containing viable and nonviable microorganism trapped in a matrix of organic matter for example exopolymers (Biofilm, 2012).
Sample drawing
Samples for microbiological analysis must always be collected in sterile glass or polypropylene bottles. After the samples were taken they must be preserved at a temperature of approximately 4 °C till they attain in the laboratory. Microorganism are affected by their environment, therefore parameters such as pH and temperature has also to be measured. (Centre d'expertise en analyse environnmentale du Québece, 2009)
General appearance
If the microorganism formed a biofilm its useful to have a look at the general appearance. Through staining the biofilm with INT, active cells can be made visible. INT is reduced to INT-formazan of an orange colour by the active electron transport. The spatial distribution of active cells can then be determined with light microscopy. This method distinguishes itself with simplicity, rapidity and the possibility to observe the biomass without preliminary treatment but has a disadvantage in a low resolution. Other methods just as scanning electron microscopy (SEM) deliver more precise data but also requires a slow and complex sample preparation (V. Lazarova et al., 1995) which includes drying and therefor deform the hydrated structure (K. Ratnayake et al. 2012).
Total biomass
Another important parameter is the total biomass it can be measured by the thicknes of a biofilm. Phyiscal methods are are light microscopy, which is a fast method but only works on thin homogenous layers, electrical conductance, which gives more precise information but only works on metallic surfaces, or or the thermal resistance. A further physical method is to weigh the dry weight, always considering that inert mass and adsorbed organic matter is also measured. Combining the data of the biofilm thickness and the dry weight, the density can be estimated. (V. Lazarova et al., 1995)
A method for measuring free-living and biofilm biomass is the standard plate count of colony forming unity (CFU). This is one of the best known microbiological techniques and is used routinely for the analysis of bacteria population. (V. Lazarova et al., 1995) To be able to count the microorganism, the sample gets elapsed on a petri dish. The single bacteria are spreaded on the medium and still growing. After an incubation time of about 18 -28 hours the single bacteria formed a colony which now can be counted (S. Sieuwerts et al., 2008) CFU has the disadvantage that only cells capable reproducing themselves are measured, which leads to an underestimation. Beside the CFU methode also spectrophotometry and turbidimetry can be used for measuring free-living biomass. (V. Lazarova et al., 1995)
Most of the descripted methods aren't capable of distinguish between different species. A brief classification can be accomplished by measuring the content of Lipopolysaccharides which are only present in the upper part of the cellular membrane of gram-negative bacteria. (V. Lazarova et al., 1995)
Biomass activity
Another important parameter for descripting a microorganism community is the biomass activity which can be determined through counting active cells or measuring different parameters such as the ATP content, the oxygen uptake rate (OUR) or the substrate removal.
Measuring the substrate removal rate is the most conventional technique to determine the microbial activity. It can be applied on free-living and fixed cells, just as the measurement of the OUR with microelectrodes.
Considering these descripted methods only some methods were applicable in the given time period and with the available material. With a day incubation time and a small material use, the CFU method would be one of those. Also the oxygen uptake rate and the evaluation of the general appearance with light microscopy should be manageable.
References:
A. Widenfalk. (2005). Interactions between Pesticides and Microorganisms in Freshwater Sediments - Toxic Effects and Implications for Bioavailability. Faculty of Natural Resources and Agricultural Sciences Department of Environmental Assessment Uppsala
Biofilm. (n.d.) Farlex Partner Medical Dictionary. (2012). Retrieved March 18 2018 from https://medical-dictionary.thefreedictionary.com/biofilm
Centre d'expertise en analyse environnmentale du Québece. (2009). Methodes for taking, preserving and analyzing samples to monitor the water quality of pools and other artificial reservoirs. Governement du Québec.
K. Ratnayake, D. C. Joyce, R. I. Webb. (2012). A convenient sample Preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage. Scientia Horticulturae 120. 12-18.
References:
A. Widenfalk. (2005). Interactions between Pesticides and Microorganisms in Freshwater Sediments - Toxic Effects and Implications for Bioavailability. Faculty of Natural Resources and Agricultural Sciences Department of Environmental Assessment Uppsala
Biofilm. (n.d.) Farlex Partner Medical Dictionary. (2012). Retrieved March 18 2018 from https://medical-dictionary.thefreedictionary.com/biofilm
Centre d'expertise en analyse environnmentale du Québece. (2009). Methodes for taking, preserving and analyzing samples to monitor the water quality of pools and other artificial reservoirs. Governement du Québec.
K. Ratnayake, D. C. Joyce, R. I. Webb. (2012). A convenient sample Preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage. Scientia Horticulturae 120. 12-18.
S. Sieuwerts, F.A.M. de Bok, E. Mols, W.M. de Vos and J.E.T. van Hylckama Vlieg. (2008). A simple and fast method for determining colony forming units. Letters in Applied Microbiology.
V. Lazarova, J. Manem. (1995). Biofilm characterization and activity analysis in water and wastewater treatment. Centre of International Research for Water and Environment (CIRSEE).
Zulauf AG. (2018). Daten und Fakten. Retrieved March 18 2018 from https://www.zulaufquelle.ch/firma/ueber-uns/daten-und-fakten/
V. Lazarova, J. Manem. (1995). Biofilm characterization and activity analysis in water and wastewater treatment. Centre of International Research for Water and Environment (CIRSEE).
Zulauf AG. (2018). Daten und Fakten. Retrieved March 18 2018 from https://www.zulaufquelle.ch/firma/ueber-uns/daten-und-fakten/
Hi Tobias,
AntwortenLöschengood overview of the situation at Zulauf nursery. I am curious, why do you think that a change in the microbial community would cause the pond to fill faster?
You seem to have concentrated your efforts on measuring bio-activity, and the quantitative evaluation of the biomass which are both important indicators but have only a limited significance by themself. Having a look at the actual composition (bacteria vs. fungi) would certainly complement the methods mentioned in your essay.
Hello Tobias,
AntwortenLöschenYou did a great job in your blog entry. I like how you solved the problem of putting the advantages and disadvantages of so many methods in such a short essay.
Which part of your question do you wan't to examine? The accumulation of the pesticides inside the irrigation system, the affect of the pesticides on the microorganism or the filling speed? I think that the question must be answered by three part steps.
Hi Tobias
AntwortenLöschenYou have described the individual methods very simply and also for laymen understandably. I think that your methods suggested at the end are suitable to answer your question. Maybe we have to define these then still somewhat more exactly, as what is done now :)
Your blog is well written and fun to read.