Characterization of terrestrial cyanobacteria to increase process efficiency in low energy consuming production processes
© Kuhne et al.; licensee Chemistry Central Ltd. 2013
Received: 22 February 2013
Accepted: 28 May 2013
Published: 12 June 2013
Terrestrial cyanobacteria have seldom been used for biotechnological processes, even though they offer great potential for new pharmaceutical products or other value-added substances. Particularly cyanobacteria of xeric habitats are of biotechnological interest, because they tolerate high temperatures, are desiccation-tolerant and feature low water consumption. In addition, the cyanobacteria collected in deserts are able to produce more photoprotective agents than their counterparts from other habitats, because of their genetical preadaptation. In this study, carotenoid and chlorophyll content of two representative terrestrial cyanobacteria strains, i.e. Nostoc muscorum and Leptolyngbya spec. sampled in Columbia (USA) and Soebatsfontein (RSA), were studied after exposure of the strains to different light conditions and cultivation temperatures. A temperature raise from 17°C to 30°C led to an increase of 46% in chlorophyll a content as well as an increase of 39% in carotenoid content of Nostoc muscorum. An irradiation raise from 19 μmol m-2s-1 to 125 μmol m-2s-1 resulted in an increase of a 10 to 20 times higher chlorophyll content. Additional results from light-curves support the potential future use of terrestrial cyanobacteria within low energy biotechnological processes using a novel type of photobioreactor to reduce the downstream process costs and nutrients needed during the cultivation. Results indicate that especially light intensity optimization currently holds unused potential.
The biotechnological utilization of cyanobacteria for the production of valuable compounds has been reported on quite often in the past. However, mainly aquatic cyanobacteria were cultivated until now, because the available fermentation technology is solely submerged . This is due to the fact, that on the one hand the organisms are easy to handle and, on the other hand, mostly marine cyanobacteria were used . Recently, terrestrial cyanobacteria have attracted more attention, because of their important ecological impact in nutrient poor arid and xeric habitats . Terrestrial cyanobacteria adapt well to abiotic environmental conditions, can withstand temperature fluctuations and low water availability , and are also capable of producing numerous biologically active- and pharmaceutical important compounds besides extracellular polymeric substances (EPS), such as Cryptophycin, the antiviral Cyanovirin-N & Scytovirin and Scytonemin [5–7].
Chlorophyll a to carotenoid ratio
It has been shown, that the light intensity has an effect not only on the chlorophyll a and carotenoid content, as well as on the composition of the different carotenoids [11, 15]. In addition, temperature is one of the most important environmental factors and plays a significant role in controlling the organisms’ activity . Therefore, the combination of these two aspects is vital in order to achieve high productivity in a potential process using terrestrial cyanobacteria.
The experiments show, that both temperature and light intensity have an effect on chlorophyll a and carotenoid content. Especially in N. muscorum a temperature rise of 13°C (from 17°C to 30°C) led to an increase of 46% of chlorophyll a and 39% of carotenoids. Interestingly, Leptolyngbya spec. shows a contrary effect. Chlorophyll a and carotenoids decrease by 86% and 68% respectively. This is due to the fact, that both organisms have different growth optima based on temperature. They can tolerate extreme temperatures  and have different mechanisms to control desiccation , but in the reported experiments they were cultivated at constant temperatures with sufficient water available to keep their metabolism active. This was done because according to first experiments their growth optima are considered to be between 20°C and 35°C.
The threefold rise in light intensity (40 μmol m-2 s-1to 125 μmol m-2 s-1) led to an unproportional increase of 10 times more chlorophyll a in N. muscorum (in comparison to 30°C) and 20 times in Leptolyngbya spec. (in comparison to 17°C). The same accounts for carotenoid contents, which increase with rising light intensities, as shown here and in other publications [11, 18, 19].
In order to calculate the carotenoid content at a light intensity of 125 μmol m-2 s-1 the chlorophyll a to carotenoid ratio (Table 1) was used. The linkage of the two components has been shown through the measured ratio during the experiments (Table 1) and by the ratio  reported, which was in the same order of magnitude. Considering this, a carotenoid content level from about 32–40 μg/g DW in N. muscorum and about 25–30 μg/g DW in Leptolyngbya spec. is assumed (24°C, 125 μmol m-2 s-1).
In exposed tropical rock habitats of Venezuela terrestrial cyanobacteria produced 553 μg/g DW chlorophyll a and 576 μg/g DW carotenoids under an extreme high natural light regime . These researchers reported light intensities of up to 2500 μmol m-2 s-1 and peaking surface temperatures of up to 50°C. The latter study and the measured light curves herein (Figure 4), show the tremendous potential in optimizing the cultivation parameters temperature and especially the light intensity. The temperature optima, though, seem to be below these peaking values. Thus, terrestrial xeric cyanobacteria can be considered interesting production organisms for carotenoid and chlorophyll, especially with regard to a low energy consuming biotechnological production process, using low light conditions.
To meet the scientific findings mentioned above and the special needs of terrestrial cyanobacteria a new photobioreactor design, especially focused on emersed cultivation was developed. A scheme of the reactor prototype is shown in Figure 1. The main parts of the reactor are glass rods, which are positioned in a modified glass-cylinder. A screw top with drillings keeps the rods in position while sealing the cylinder. Light is applied via LEDs positioned above the reactor and nutrients, as well as, water are supplemented as aerosol. Additional installations to the glass-cylinder allow gas control and gas exchange measurements. At the bottom of the reactor the accumulated medium can be recycled. Cells that could be potentially removed by washing are restrained by a micro strainer. Different phototrophic organisms can be cultivated on the surface of the glass rods, where light is applied to them. This special design features various advantages in comparison to classical photobioreactors, such as the possibility of humidity and irradiation regulation, as well as water on hand, for organisms. It is possible to cultivate terrestrial cyanobacteria under controlled and reproducible conditions. Moreover, natural conditions such as circadian rhythms and even humidity or temperature change over day can be imitated. In comparison to outdoor cultivation numbering up and sterility make the photobioreactor ideal for screening desiccation tolerant organisms in general, for example for pharmaceutical processes. Due to medium recycling, the amount of water needed for cultivation and occurring waste can be reduced. Even more importantly, the energy needed for the downstream process can be reduced significantly, because the cells can be dried and retrieved inside the photobioreactor. No further purification, centrifugation and drying is needed. Together with the elimination of energy needed for mixing up to 40 percent of the process costs can potentially be saved [20, 21]. Kieseler et al.  recently pointed out, that about 15 percent resulting algae biomass is needed only to cover the drying costs, which can be avoided using our reactor design ether. With a low required energy-input the reactor also features low energy consuming production and screening demands of nowadays and thus is applicable within further studies considering terrestrial cyanobacteria. An additional publication featuring further details of the new photobioreactor is currently in preparation.
The terrestrial cyanobacteria Nostoc muscorum (PCC 7906) and Leptolyngbya spec. (BB 2292(5)1) isolated from xeric habitats on soil in Columbia (USA) and Soebatsfontein (RSA) respectively, were cultivated both under the same light conditions (40 μmol m-2 s-1) at different temperatures (17°C and 30°C) and with the same temperature (24°C) but at different low light conditions (19 μmol m-2 s-1 and 125 μmol m-2 s-1). The response to light was analyzed with rapid light response curves, determined by chlorophyll a fluorescence. Photosynthetic saturation between photosynthetic flux densities of 600 and 800 μmol m-2s-1 was revealed. This indicates that the strains were investigated while they were still in the linear increase of light limitation at the applied light intensities. The cultivation took place in temperature controlled photo incubators (program-controlled Incubator, Type KBP, Series 2000, Tritec Hannover, D) for 4 weeks and liquid cyanobacteria medium BG11 was used. The temperature and humidity was measured with miniaturized data logger (i-Buttons, Maxim Integrates products, Inc., Sunnywvale, CA, USA). The duration of light exposure was 14/10 hrs. and the light intensities were measured with a quantum sensor (Licor 190A, Licor-Biosciences Lincoln, NE, USA).
The cultures were homogenized using glass balls (0,5 mm diameter) and a swing mill (Retsch MM200; Retsch GmbH, Haan, D) for 90 sec at a frequency of 20 s-1. The dry weight was determined using a volume of 500 μL of the homogenized culture for centrifugation at 10,600g and drying afterwards at 60°C. The optical density (OD) was measured at a wavelength of 665 nm (OD665) in 96-well micro plates (PS-Micro plate, 96-well, colorless; Greiner Bio-One GmbH, Frickenhausen, D) using a micro plate Spectrophotometer (Epoch micro plate, BioTek Germany, Bad Friedrichshall, D). Both parameters were correlated to pigment contents to calculate the chlorophyll a and carotenoid content on a dry weight basis in [μg/g DW].
It has been shown, that terrestrial cyanobacteria feature interesting properties regarding biotechnological production processes. Due to their genetical preadaptation to light, temperature and desiccation stress cyanobacteria are less sensitive to fluctuations regarding these parameters during production processes. Because of their adaption to high irradiation, a photoinhibition which is often considered a problem during the cultivation of phototrophic organisms, is far less likely and their desiccation tolerance makes them ideal for low energy consuming processes. This is due to the fact, that less water and medium itself is needed during the cultivation process and direct lighting via sunlight (after a possible upscale) can be used more easily. Our data shows the tremendous influence of light and irradiance on the formation of this exemplary products, whereas the temperature plays a minor role. As shown in this paper, a special photobioreactor design for emersed cultivation of phototrophic organisms, can contribute further to the optimization of a biotechnological process using terrestrial cyanobacteria. To make a future industrial application more likely the costs related to the downstream process can be reduced significantly because no energy is needed for mixing, centrifugation, purification or drying and less waste is created. That leads to a process, which is much more sustainable than already existing bioprocesses and especially than comparable chemical ones.
This preliminary study draws attention to a currently underexploited group of microorganisms and shows first data regarding possible cultivation parameters. By applying this novel type of photobioreactor this resource can efficiently be tapped for several biotechnological production processes.
This research was supported by the Deutsche Forschungsgemeinschaft (UL 170/7-1, LA 1026/9-1). The research team would like to thank Sandra Meck and Christian Seebach for their assistance during the experiments.
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