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Stop The Green Biofilm: Studies On Growth And Adaptation Of Lampenflora Under Led Light In Showcaves Печать E-mail
07.12.2014 01:47

U. Peters, L. Groos, F. Schäfer
Zeitsprünge e.V.; Breitscheid, Germany

Caves have always had special attraction to mankind. In the last few years, the awareness that these biotopes are worthy of protection has grown substantially. Most show cave operators have recognized the importance of sustainable working within these unique natural habitats. Visitor expectations are more than the mere feeling of being inside a cave and feeling the unique atmosphere of it. Today, to be successful in the show cave market, operators are required to fully illuminate the cave, include presentations, events and special effects. However, all these man-made influences have a negative impact on the natural balance of the cave. This article will focus on the aspect of artificial growth of algae in caves- defined as ‘Lampenflora’. In the last decades the intensity of illumination has increased – often by using high performance halogen lamps. To reduce issues with “Lampenflora” many commercially operated caves have changed the light source to LED. In the EU, the directive 2005/32/EG prohibits the use of normal light bulbs. One option is the installation of LED-Light. The advantages are higher light yield, lower energy costs, longer lifespan, and a spectrum which may reduce or even avoid ”Lampenflora”. The latter aspect is not yet scientifically proven and there remain a considerable number of unanswered questions: What would happen with existing biofilms when changing the lighting? Are there long-term adaption processes? Can algae grow in newly developed LED-illuminated caves? What conceivable con- cepts exist for long- term application to prevent “Lampenflora”?

The starting point for our studies are the newly developed show cave called “Herbst- labyrinth” in central Germany, Breitscheid and the investigations by the Karst-Research-Institute of Postojna. The institute provides extensive data on the natural cave algal colonization [6, 7] where the first experiments with Chlorella algae under LED light were performed [8].
Initially, we used an experimental setup similar to the Postojna Institute. Our research also concentrated on Chlorella vulgaris. We performed unprecedented analysis of the growth pattern of cyanobacteria Synechocystis spec. under laboratory conditions. Cyanobacteria are the primary organism in succession [6], therefore very important for the aspects of Lampenflora.
Secondly, we tested the viability of these algae cultures under the light conditions similar to those found in the cave.
The next step documented the transformation of the biofilm in two German show caves (Schillat-cave; Iberg-cave) following the in- stallation of LED light. We attempted to determine if there are adaption processes in the photosynthetic pigments as a result of the modified light spectrum.
Material and Methods. growth-ex- periments under laboratory conditions: For the algae culture experiments, sam- ples of Chlorella vulgaris (#211-12) and Synechocystis spec. (#92.72) were pur- chased at the EPSAG in Gottingen, Germany. As the nutrient we used “KUHL”-Medium (green algae) and the “BG-11”-Medium (cyanobacteria) (EPSAG).

Fig. 1. Emission spectrum of the different used light sources

After allowing the algae cultures to adapt to the low light conditions (50 µmol photons/m2s) and cave temperature (10-12° C) in a similar cave-like environment, our process com- menced with a defined algae concentration of 1-2 x 107 organism per 100 ml which corresponded to a photometric absorption (436 nm) of 0,01. For comparison, we used a standard halogen lamp (Osram, Halopar 20W), a LED from the GermTec Company (Herborn, Germany) and an aquarist lamp with an ideal photosynthetic spectrum (AquaGlo, Hagen Deutschland GmbH). The light intensity was adjusted to both 10 and 30 µmol photons/m2s (PAR 10 or PAR 30) at 8h light per day and tested with 3 parallel samples for each species. Samples were taken twice per week measuring the con- centration at 436 nm over a period of 6 weeks (TOM – time of measurement).
Growth-experiments in the showcave ‘herbstlabyrinth’: In September 2012 observations started on the growth behavior of both types of algae in the showcave “Herbstlabyrinth”. Given the experience of the first studies, it was decided to minimize the taking of samples and to close the Erlenmeyer-flasks almost completely. These measures were taken to avoid contamination of the samples in the ‘Herbstlabyrinth’. The algae cultures were exposed to 200 minutes of light per week with an intensity around 10 µmol photons/m2s. Measurements took place on a monthly basis until January 2013, with a final detection in January 2014.
Observations of the alteration of the biofilms in two different show caves: In two show caves in Germany it was now possible to witness the alteration of the biofilms following the re- placement of the light bulbs to LED lights. The first season utilizing the new lighting system was started in both the Schillat and Iberger cave in the spring of 2013. The Schillat cave is located in Hessisch-Oldendorf near Hannover and is the most northern CaCO3 dripstone cave in Germany; the Iberger-cave belongs to the community of Bad Grund in the mountains of Harz.
Samples were taken from the biofilm at different locations in each cave and checked against the entire absorbance spectrum (400-750 nm). Samples were prepared for photometric measurement after the following scheme (Tab. 1).

Table 1: Procedure of the extraction for the photometric measurement
• 1 ml sampling into an Eppendorf-tube
• Centrifuge 60s at 7500 g
• Pipette of the entire liquid
• Add 100µl dH2O
• Give into solution
• Add 900µl methanol
• Store 30 min in a refrigerator
• Centrifuge 60 s at 7000 g

Samples were taken in April 2013 and in October 2013. A specific difficulty occurring re- peatedly was with the small amount of drip- stone ground algae, which required to be sampled without contamination. As a result, the algae quantity decreased in all places. In order to compare the data, a mathematical adjust- ment was applied in the range of the approxi- mate linear relationship of the photometry (Fig. 5a-d).
Statistical analysis: To determine whether the measured differences are statistically significant, the rank correlation test of Wilcoxon, Mann and Whitney (U-test/Sachs 1984) was applied.
Results. Growth-experiments under laboratory conditions: Considering the maximum differences of the absorption during the experimental phase (TOM 1-12), a clear difference between the cyanobacteria and green algae can be detected (Fig. 2).

Fig. 2. max. differences of the extinction over a 6 week growing period under different light intension (PAR 10 & 30)

Chlorella can live and grow under all three light spectra. The growth rate under PAR 10 can reach approximately 33 - 50 % compared to PAR 30 conditions.
Synechocystis, however, only grows significantly under the lighting conditions of the Aqua-Glo PAR 30. Under the PAR 10 lighting it reaches only a tenth of the growth rate under the AquaGlo
PAR 30.
Under LED light PAR 30 no significant growth could be observed. The absorption was in the range of AquaGlo PAR 10 and after six weeks almost corresponded with the algae concentration at the beginning of the experiment. In addition, under LED PAR 10 there is also decrease in cell density in the culture. A similar outcome applies to the experimental cultures under halogen light.
The significance matrix (Fig. 3) demonstrates that the results shown in Fig. 2 have a discernible difference at a significance level of P ? 0.02 which is represented in the diagram.

Growth-experiments in the show-cave ‘herbstlabyrinth’: The extraordinary result of the long-term experiments (more than one year) was the survival of the Chlorella. However, it appeared that Synechocystis was unable to survive. It was interesting to note that the concentration increased during the first 4 months. The shape of the graph after 4 months is indeterminable due to the missing data between the two last measurements.

Fig. 3. significance matrix’ for the culture experiments:
‘-‘ no significance;
‘+’ significance level P≤0.02,
white – missing data

The results show that we require to be patient to see a transformation process in the biofilm of Lampenflora.
Observation of the alteration of the biofilms in two different showcaves: The re- sults of the full spectrum analyses (Fig. 5) show different variations inside the range of the 400 – 650 nm wavelength after half a year. There is less or no variation at the red-light-maximum of chlorophylla (662 nm). For that reason, adjust- ments were made to the different photometric curves on this maximum. We have identified differ- ent absorption rates for different biofilms. Fig. 5a and 5c demonstrate typical characteristics identified for biofilms containing a majority of green algae. Fig. 5b represents the results from a cave biofilm with a high density of Xanthophyceae and green algae.
The absorption in the range of 440 – 490 nm (carotenoid shoulder) was higher at the second TOM. Also a higher absorption is detected in the emission spectrum of the LED-light (500-600 nm). Fig. 5d describes the behavior of a biofilm which is dominated by cyanobacteria. In contrast to Fig. 5a – 5c, the second sample has a lower blue-light absorption and the high- est increase of absorption at the wavelength between (550 – 650 nm). The maximum absorptions of the antenna pigments are found at this wavelength.

Fig. 4. course of algal growth during the growth-experiment in the showcave ‘Herbstlabyrinth’ – 200min light/week – each point is a measurement – dashed line represents estimated course over nine month period

Fig. 5. full spectrum analysis of algae samples of two German showcaves: a and b are from Iberger Showcave; c and d are from the Schillatcave. The blue line represents the ?rst measurement (April 2013) and the green line the latest one (October 2013). The blue arrows mark the conspicuous changing of carotenoids and antenna pigments. The Samples of a and c were dominated by green algae; d by cyanobacteria; b by a mixture of diatoms and cyanobacteria.

Discussion. The last few years have revealed a growing interest for the sustainable use of show caves, with focused on the negative impact of the commercial operation of caves now in the spotlight of scientific research [4]. In order to run an environmental friendly and sustainable show cave, the consideration of scientific facts is imperative. To date, little is known about the growth conditions and behavior of Lampenflora.
In early study results dating back to the 1970’s, Planina (1974) sets the minimum light requirements to 100 h/y. In studies performed by Rajczy (1989) investigating the compensation point, he suggests a longer distance between light source and dripstone surface. Mulec et al. (2008) investigated the minimum light intensity of aerobic cave algae colonies in natural habitats and registered a result of 0,06 µmol photons/m2s.
At an international conference on cave lighting in Budapest in 2002, Olson published the first results of research performed using LED lamps with an emission maximum at 595 nm. After purging, a re-colonization with algae under these conditions could be prevented for 1.5 years. A similar outcome has been noted in the Bad Segeberg cave (statement - VdHK - A. Ipsen 2012). A proliferation of green algae, diatoms and cyanobacteria is described in the literature of Jakob (1997), Rosenkranz (2008), Anderson and McIntosh (1991).
The focus of our present research concerns the parameter constellations that may affect Lampenflora.
Therefore, the aim is to identify a scientifically proven solution for the long term prevention of the growth Lampenflora. An urgent solution is required, as the directive 2009/125/EG forces showcave operators to discard traditional lighting and invest in low energy light sources.
The combined investments of Germany’s 53 show caves add up to millions of Euros.
The opportunity to introduce a sustainable light concept is complicated by the lack of scientific data.
The working group of Janez Mulec has done most of the research on this field from the Karst Research Institute of Postojna (2008, 2009, 2014).
Our investigations have revealed data on the survival rates of two groups of algae under different conditions, leading to an assumption that there is an adaption process in the natural biofilm after changing to LED light.
Chlorella: The test subject Chlorella vulgaris is a typical representative of Chlorophyta in Lampenflora biofilms [7]. The initial hypothesis that algae survival was least probable under LED light conditions was confirmed. However, within the experimental period, an increase in biomass could be identified. These results match the findings at Postojna Institute [8].
In contrast, the green algae in the “Herbstlabyrinth” demonstrated a sharp decline in vitality only after the period of one year. However, it should be taken into consideration that the light supply ranged around 200h/ year, which doubles the light minimum set by Planina (1974), whilst the algae had optimal nutrient supply.
Synechocystis: The category of Cyanobacteria including the genus Synechocystis represents the greatest biodiversity in caves. These organisms are able to survive with less light with a com- pensation point ranging from 5 µmol photons/m2s down to 0,11 µmol photons/m2s [5, 7].
The findings are the first experimental data collected of the genus Synechocystis under LED- light conditions. To the best of our knowledge, to date, no comparable data exists. The findings are unusual considering the reported behavior of Synechocystis in natural habitats [5]. The observed decline in vitality under PAR 10 LED light in the Synechocystis culture indicates that the organisms are unable to survive permanently under the test conditions. The results from “Herbstlabyrinth” confirm the first analysis. After one year, under the test conditions, there was no measurable absorption. The interpretation of the results is difficult, as the long term storage of Synechocystis is complicated (pers. Mess. EPSAG). Otherwise, Mulec and Kosi (2008) reported a periodical lack of Cyanobacteria building new biofilms. Therefore, it is unclear if the observed widespread death is due to the effect of the light spectrum or if there are unknown factors which are yet to be discovered.
After the period of one year under the LED light, the data from the show caves indicated a strong decline, however no extinction (VdHK – Anne Ipsen 2012).
Alteration of the absorption spectrum: The results of the investigation have shown that the use of LED light may be an appropriate method to change and reduce the Lampenflora. In order to interpret the findings of the algae culture experiments, it is necessary to take into account the special conditions:
a) Monocultures i.e. no competitors
b) Optimal species-specific nutrient supply
Therefore, we would question whether the algae are able to survive in their respective cave habitats, and if they are able to colonize a new cave habitat under LED light conditions. Given that the “Herbstlabyrinth” is free of algae, may be an indicator confirming this hypothesis.
The next step determined what effects a change to LED light would have on the already existing Lampenflora. A reduction of Lampenflora was identified in both show caves (Iberger Hohle, Schillathohle) as well as in “Bad Segeberg” [2].
What changes does the reduced biofilm undergo?
The interpretation of our measurements leads to two possible outcomes. There may be a change of the biofilm composition e.g. a shift in the proportion between cyanobacteria and green algae. On the other hand, it is possible that adaptation processes might begin. Such adaptation processes are widely known; especially for the antenna pigments of the cyanobacteria [14]. The differences between the samples are very conspicuous. Currently, no clear pattern has been detected in the changes of the spectra.
However, it may be possible to identify a trend out with the data. Based on the identification of species in the samples provided by EPSAG, the absorption spectra can be assigned to different groups of algae, i.e. green algae, cyanobacteria and xanthophyceae. A higher absorption in the wavelength of phycobilisomes (Phycoerythrin, Phycocyanin, Allophycocyanin) was detected when the sample was dominated by cyanobacteria. A conspicuous change was identified in the maximum absorption wavelength of carotenoids in samples dominated by green algae and Xanthophyceae.
Similar results were published by Mulec (2014), displaying a typical full spectrum analysis of Chlorella vulgaris cultures growing under different light conditions.
Final clarity can only be provided by a higher density of data. It is important to identify the leading process in order to establish a sustainable light concept for caves.
Conclusion – relevance of the results in relation to Cave protection. Our investigation has shown that both Chlorella vulgaris and Synechocystis spec. are capable of limited survival. However, under the condition of LED illuminated caves they are at the verge of extinction. Existent biofilms are reduced after changing the light sources to LED; however physiological adaptation processes would start. A holistic approach strategy of changing the light source to LED is not enough. Use of LED light with the lowest possible PAR (not more than 30 µmol photons/m2s at the surface of the speleothems) requires the addition of:
1. Absolute avoidance of nutrient inputs on speleothems
2. Intensive monitoring of vulnerable areas for the early detection of biofilm
3. If possible, a concept of alternating light to reduce the time of illumination – it may therefore be useful to repeat the studies of Planina (1974) to determine the maximum time of LED illumination (under different PAR regime) in order to reduce Lampenflora effectively.
Acknowledgements. We would like to thank Janez Mulec for literature support. The coopera- tion with Maike Lorenz from EPSAG, Gottingen was a pleasure, especially for determination of algae in the biofilm samples. We thank Benjamin Eisenhardt (AG Prof Dr. Klug, University Gie?en) for analytic support. There is also a huge thanks going out to the company GermTec Herborn for providing the LED-lamps. Without the assistance of Anke Peters it would not have been possible to collect and analyze all the samples. At least the authors are grateful to Leonard Gerz for revision of the English text.

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15. Nutrient media:

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