Curr Microbiol (2014) 69:699–702 DOI 10.1007/s00284-014-0645-1

Biofilm Formation by Chlorella vulgaris is Affected by Light Quality Malin Hultberg • Ha˚kan Asp • Salla Marttila • Karl-Johan Bergstrand • Susanne Gustafsson

Received: 13 February 2014 / Accepted: 9 May 2014 / Published online: 2 July 2014 Ó Springer Science+Business Media New York 2014

Abstract Formation of biofilm on surfaces is a common feature in aquatic environments. Major groups of inhabitants in conditions where light is present are photoautotrophic microorganisms, such as cyanobacteria and microalgae. This study examined the effect of light quality on growth and biofilm formation of the microalgal species Chlorella vulgaris. Dense biofilm formation and aggregated growth of cells were observed in treatments exposed to blue, purple and white light. Less dense biofilm formation and solitary growth of cells were observed in treatments exposed to red, yellow or green light. Microalgal biofilms are of high importance in many respects, not least from an economic perspective. One example is the intense efforts undertaken to control biofilm formation on technical surfaces such as ship hulls. The present study suggests that light quality plays a role in biofilm formation and that blue-light receptors may be involved.

Introduction The intensity, duration and quality of the light available are major factors regulating growth of photosynthetic organisms such as microalgae. Wavelengths of 450–475 and

M. Hultberg (&)  H. Asp  K.-J. Bergstrand Department of Biosystems and Technology, Swedish University of Agricultural Sciences, P.O. Box 103, 230 53 Alnarp, Sweden e-mail: [email protected] S. Marttila Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden S. Gustafsson Ekoll AB, Malmo¨, Sweden

630–675 nm are to a high extent absorbed by the chlorophylls, the main pigments responsible for harvesting solar energy on earth [9]. However, in addition to major pigments such as chlorophylls, phycobilins and carotenoids, there are a wide range of photoreceptors which react to the quality of the light, affecting the development of the microalgae [8]. For plants, a vast amount of research has been performed in this area and it is well-known that light quality plays a major signalling role in plant development [1, 3, 6, 17]. However, less is known about the effects of light quality on photosynthetic microorganisms. Formation of a biofilm, the initial step in biofouling, is a common feature on surfaces in aquatic environments [2]. A major group of inhabitants in the initial biofilm formed under conditions where light is present, phototrophic biofilms, are cyanobacteria and microalgae [12, 14]. There are many practical issues arising from phototrophic biofilm formation, including problems with biofouling on technical surfaces such as ship hulls and lowered light transmittance in photobioreactors [7, 14]. Recent research shows that bacteria, both phototrophs and chemotrophs, can use the quality of the light available to make important lifestyle decisions such as persistence in a single cell state or as a surface-attached biofilm [5]. Also changing the quality of the light used for illumination, from white light to green light, has been shown to affect the phototropic biofilms developing on illuminated work of arts [16]. In the present study, we examined the effect of light quality on biofilm formation by the green algal species Chlorella vulgaris. This well-studied microalgal species has been shown to be one of the dominant microorganisms in the early development of biofilms in industrial systems [18]. We also examined the amount of bacteria in the medium and the pH, as these factors have previously been reported to affect biofilm formation [18].



M. Hultberg et al.: Biofilm by C. vulgaris and Light Quality

Materials and Methods Microorganisms The microalgae C. vulgaris 211/11B, obtained from The culture collection of algae and protozoa (CCAP), Oban, UK, was used in the experiments. The strain was routinely cultured in Z8, a standard medium for green algae [11]. Experimental Set-Up In the experiments, a light–dark photoperiod of 16–8 h and a light intensity of 100 lmol m-2 s-1 were used. Six different colours of light emitting diodes (LED), providing narrow-band light of different wavelengths, were applied: blue (460 nm), green (525 nm), yellow (585 nm), red (620 nm), purple (eight parts 660 nm and one part 460 nm) and white (430–730 nm). In order to avoid mixing with other wavelength sources, the LED treatments were separated by plastic coloured black on the outside and white on the inside for maximum reflection. The temperature in the chamber was set to 20 °C. The experiment was performed as batch cultures with a volume of 50 mL Z8 [11] in 250 mL Erlenmeyer glass flasks. The start density was 104 cells mL-1 and the experiment was ended after 7 days. Analysis After 4, 6 and 7 days, samples comprising of cover glass and medium were removed from the different treatments and examined under light microscope (Nikon Eclipse 50i, Japan). After 7 days, the biofilm formation on the glass surface was studied by scanning electron microscopy (SEM). For this purpose, biofilm on small pieces of glass was fixed overnight in 2.5 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) at 4 °C. After washing three times with phosphate buffer, the specimens were dehydrated through an ethanol series (10, 30, 50, 70, 96 and 99.5 %) and inserted into a critical point dryer (Balzers CPD 020, Balzers, Hudson, USA). The dried samples were mounted on specimen stubs with double-sided tape. The stubs were covered with gold/palladium (3:2) in a JEOL JFC-1100 ion sputter (JEOL, Tokyo, Japan) and examined in a LEO 435 VP scanning electron microscope (LEO, Cambridge, UK). Photographs were taken to visualize differences in the appearance of the biofilm between treatments. In order to separate the biomass in the liquid phase from that in biofilm, the medium was poured off and centrifuged at 3,0009g (Avanti J-20, Beckman Coulter, CA, USA) for 20 min and the pellet was collected. After this step, the biomass adhering to the glass was collected. The


Fig. 1 Biofilm formation by Chlorella vulgaris after 7 days of algal growth in the treatments with blue and red light. In terms of visual appearance, the treatments where blue, purple and white light were used resulted in high biofilm formation, whereas the treatments with red, yellow and green light resulted in less biofilm formation

Erlenmeyer flasks were carefully washed with 10 mL sterile water in order to remove loosely adhering cells. After this step, another 10 mL sterile water were added and the biofilm was removed with a cell scraper (Naige Nunc International, USA). The biomass was collected through centrifugation as described above. All samples were lyophilized and the dry weight was recorded. In order to study the amount of bacteria in the different treatments, viable counts were performed after 7 days. Aliquots of the medium were diluted stepwise and inoculated into R2A Agar (Difco). The petri dishes were incubated at 20 °C for 72 h and dishes with a colony count between 30 and 300 were used for enumeration. The pH of the medium after 7 days of growth was also measured. Statistics Each experiment was carried out in triplicate and mean values and associated standard deviation are reported. The whole experiment was repeated. The data were analysed by analysis of variance followed by Tukey’s multiple comparison test and differences were considered significant at P \ 0.05 (Minitab, version 16).

M. Hultberg et al.: Biofilm by C. vulgaris and Light Quality


Table 1 Amount of biomass (mg DW L-1) in medium and in biofilm, and percentage of total biomass in biofilm, after 7 days of growth of Chlorella vulgaris under different light quality (mean ± SD, n = 3) Light quality White

Biomass in medium (mg DW L-1)

Biomass in biofilm (mg DW L-1)

Total biomass (mg DW L-1)

Percentage of total biomass in biofilm

81.1 ± 20.2a

406.7 ± 20.8a

487.7 ± 26.5a

83.4 %


210.5 ± 17.0b

34.3 ± 12.5b

236.7 ± 20.6b

14.0 %

Yellow Blue

213.3 ± 32.1b 83.3 ± 32.1a

36.7 ± 15.3b 367.0 ± 40.5a

250.0 ± 40.0b 450.7 ± 69.0a

14.7 % 81.5 %


69.2 ± 12.3a

333.4 ± 84.3a

403.0 ± 69.1a

82.8 %


203.2 ± 35.1b

56.7 ± 35.1b

260.0 ± 40.0b

21.8 %

Values within columns followed by different letters are significantly different (P \ 0.05, Tukey’s test) DW dry weight

Results and Discussion There was a clear visual difference in biofilm formation between the different treatments by the end of the experiment (Fig. 1). In the treatments exposed to red, yellow and green light, it was evident that less biofilm had formed after 7 days of algal growth compared with in the treatments exposed to blue, purple and white light (Fig. 1). The amount of bacteria in the medium varied between log 5.0– log 4.8 cfu mL-1 and no significant differences were found between the different treatments (P = 0.221). Furthermore, there were no significant differences in pH of the medium, which varied between 9.25 and 9.72 (P = 0.075). When the biomass suspended in the medium and that present in the biofilm was measured, significant differences were found between the treatments. The biomass in the biofilm, expressed as percentage of total biomass, varied from 81.5 to 83.4 % for the blue, purple and white light treatments, whereas it varied from 14.0 to 21.8 % for the red, yellow and green light treatments (Table 1). When glass surfaces from the different treatments were examined by light microscopy, the treatments exposed to blue, purple or white light showed the presence of biofilm formation already after 4 days of growth. This was not observed for the other treatments. Light microscopy analysis of the cells in the medium showed that the cells grown in blue, purple or white light were more often aggregated into large groups, whereas the cells grown in red, yellow and green light were mostly present in a solitary state (Fig. 2). This difference between the treatments was evident already after 4 days of algal growth. Light quality has been reported to have an impact on production of extracellular polysaccharide in microalgae [21] and this potentially could explain this result. Scanning electron microscopy of the glass surfaces after 7 days of growth confirmed the finding of a different distribution of the biomass (Fig. 3). Biofilm formation was clearly apparent in the blue-light samples, whereas

Fig. 2 Cells of Chlorella vulgaris after 7 days of algal growth in the treatments with blue and red light. Cells grown in blue (and purple or white) light were more highly aggregated into large groups, whereas the cells grown in red (or yellow and green) light were mostly present in a solitary state. Scale bar 10 lm

the red-light samples had hardly any biofilm formation except for occasional minor groups of the algae. Red light is commonly suggested as optimal for biomass productivity in C. vulgaris, since this microalga has a high concentration of chlorophyll and hence absorbs efficiently in the red wavelength area [4]. This has also been confirmed in studies where C. vulgaris has been grown planktonic [20]. In the present study, the total amount of biomass was significantly higher in the treatments where biofilm formation was observed. This result suggests that growth in biofilm might be favourable for biomass production. Biofilm formation was observed in the treatments receiving either blue light only or white and purple light, which contain parts of blue light. This suggests that it was the blue light that stimulated biofilm formation. From studies on prokaryotes it is known that different classes of blue-light receptors can be involved in biofilm formation [5]. These studies have shown that the response is not uniform among the prokaryotes, with both blue-light activated biofilm formation [13] and blue-light inhibition of biofilm formation [10] having been observed.



M. Hultberg et al.: Biofilm by C. vulgaris and Light Quality

Fig. 3 Scanning electron micrographs of the biofilm formed by Chlorella vulgaris after 7 days of algal growth in the treatments with blue and red light. There was dense colonization of the glass surface by microalgae when blue light was used for cultivation and only occasional grouping when red light was used. Scale bar 1 lm

The importance of light for adhesion of microalgae to a surface and thereby formation of a biofilm has only been reported in few studies [16]. There are, however, major implications of this finding. Considering the practical problems encountered due to biofilm formation, the potential for using light as a tool for managing biofilm formation is interesting. For example, light provided by LEDs could be used in designing systems with a low tendency for biofilm formation. Another interesting application of these findings would be manipulation of light quality for harvest of microalgae. A bottleneck in the development of new algal culturing systems is the harvest methods existing today, filtering, chemical flocculation and centrifugation, which are both expensive and energydemanding [19]. Light could potentially be used for stimulating growth as a biofilm with the advantages of an easy harvest as discussed by Roeselers et al. [15]. Acknowledgments The authors thank Kerstin Brismar for skilful ˚ ngpannefo¨reningen help with SEM. The project was financed by the A Foundation for Research and Development.

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Biofilm formation by Chlorella vulgaris is affected by light quality.

Formation of biofilm on surfaces is a common feature in aquatic environments. Major groups of inhabitants in conditions where light is present are pho...
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