J Appl Phycol DOI 10.1007/s10811-014-0436-x

Comparative analysis of growth and carotenoid accumulation of arborum in aerial, subaerial, and aquatic cultivation

Lin Chen & Lanlan Zhang & Wei Zhang & Tianzhong Liu

Received: 17 June 2014 /Revised and accepted: 30 September 2014 # Springer Science+Business Media Dordrecht 2014

Abstract The aerial filamentous microalga, Trentepohlia Introduction arborum (), was cultured in three habitats - an aerial, a subaerial and an aquatic one - using different types of The algal genus Trentepohlia is widespread in different loca- bioreactors. The growth, carotenoid productivity, and morpho- tions such as tropical, subtropical, and temperate regions, even logic differences of T. arborum in different habitats were inves- in frigid zones (Allali et al. 2013;Nashetal.1987; Jorgensen tigated. The maximum specific growth rate (μ)ofthealga and Tonsberg 1988; Liu et al. 2012). The colonies of obtained in the logarithmic phase in the subaerial habitat was Trentepohlia generally grow on the surface of rocks, soil, 0.034 h−1. HPLC analysis also demonstrated that T. arborum walls, or tree trunks, to form a spectacular orange or red accumulated a high content of carotenoids. Zeaxanthin was the “carpet” named “Red stone” (Liu et al. 2012), because it is primary carotenoid in subaerial culture, while β,β-carotene was rich in carotenoid content, especially β-carotene (Kjosen et al. dominant in aerial culture. The maximum carotenoid produc- 1972; Czeczuga and Maximov 1996; Abe et al. 1998; tivity, 67.7 mg m−2 day−1, was reached when T. arborum was Mukherjee et al. 2010). Moreover, some carotenoids, unique cultured in the subaerial habitat under nitrogen depletion using to this group, have been found in Trentepohlia gobii a novel attached cultivation bioreactor. Overall, it was demon- Meyer (Czeczuga and Maximov 1996)andTrentepohlia strated that T. arborum exhibited a relatively high growth rate jolithus (L.) Wallroth (Kjosen et al. 1972;Nybraateand and carotenoid productivity under attached cultivation. Liaaen-Jensen 1974). In addition to carotenoids, Trentepohlia also produces some antioxidant and antimicrobially active substances such as phenolic and Keywords β-Carotene . Trentepohlia arborum . Attached flavonoid compounds (Simic et al. 2012). Therefore, this cultivation . Aerial alga has potential economic importance. Many studies have been carried out on Trentepohlia spp., most of them focused mainly on (Lopez-Bautista et al. 2006; Rindi and Lopez-Bautista 2007; Rindi et al. 2009), ecology (Rindi and Guiry 2002; Rindi and Lopez-Bautista L. Chen : L. Zhang : W. Zhang : T. Liu (*) Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and 2007; Liu et al. 2012; Allali et al. 2013), and physiological Bioprocess Technology, Chinese Academy of Sciences, characteristics (Abe et al. 1999; Chapman et al. 2001; Gupta Qingdao 266101, Shandong, People’s Republic of China and Agrawal 2004; Mukherjee et al. 2010). Only a few at- e-mail: [email protected] tempts have been made to culture Trentepohlia odorata (F.H. L. Chen Wiggers) Wittrock (Ho et al. 1983; Lee et al. 1990)and e-mail: [email protected] (L.) C.F.P. Martius (Abe et al. 1998; L. Zhang 2003; 2008) for biotechnological applications such as biolog- College of Food Science and Engineering, Ocean University of ical nitrogen removal. China, Qingdao 266003, People’s Republic of China Trentepohlia is referred to as an aerial (Abe et al. 1998)or subaerial genus (Ho et al. 1983; Rindi and Guiry 2002;Liu L. Chen University of Chinese Academy of Sciences, Beijing 100049, et al. 2012), with none ever found in natural aquatic habitats People’sRepublicofChina (Lopez-Bautista et al. 2002). However, some species, such as J Appl Phycol

T. aurea,canbegrownsuccessfullyinliquidmediuminthe was dripped down onto the filter paper (Fig. 1d)througha laboratory (Abe et al. 1998). However, the relatively slow perforated nylon tubing positioned between the filter paper growth encountered in liquid-suspended cultures is unsuit- and glass plate at the top of the glass chamber, using a able to produce carotenoids for industrial purposes. It mini peristaltic pump. In the second type of reactor, the should also be noted, however, that some species of medium was spread onto the algae cells on the surface of Trentepohlia alternate between periods of exposure to air the filter paper using a mini ultrasonic atomizer (HL- and immersion by dew and rain in their natural habitats. MM001, Zhongshan Hongling Electrical Appliance Co., Therefore, Trentepohlia exhibits the ability to survive in Ltd., China), which was placed at the bottom of the cham- both aquatic and aerial conditions. Unlike other species of ber and was immerged in the liquid medium. The third Trentepohlia, T. arborum (C. Agardh) Hariot has been type of reactor was a conventional air-bubbling column reported growing very vigorously in natural habitats (Rindi with 3-cm diameter and 50-cm height (Fig. 1b). As a result, et al. 2005). However, there are no reports on its cultiva- the algae T. arborum was grown in three different artificial tion and product accumulation. habitats of subaerial (Fig. 1a), aerial (Fig. 1c), and aquatic In the present study, alga T. arborum was cultured in three (Fig. 1b)conditions. types of artificial habitats, namely, aquatic, subaerial, and For subaerial and aerial cultivation, the algal inoculum aerial, to study growth and carotenoid accumulation. Morpho- was evenly filtered onto a cellulose filter paper, which logical differences in the varying cultivation modes were also showed superior performance for algae growth compared compared. to glass fiber (GF), nitrocellulose (NC), and cellulose filter papers in preliminary experiments, to form an algal “disk” with a footprint of 10 cm2. The inoculum density Materials and methods of the algal disk was controlled at about 5 mg (dry weight) by adjusting the volume of the prepared algal The alga T. arborum was a gift of Prof. Liu Guo-Xiang from broth. Then, the algal “disk” wasplacedonthesurface the Institute of Hydrobiology, Chinese Academy of Sciences. of a glass plate, and finally, the device was placed into the The strain was isolated from the bark of a Ficus virens in chambers for attached cultivation with a constant medium Sichuan, China. The strain was identified by morphology and supply of 60 mL h−1 via the peristaltic pump or the SSU rDNA gene sequence comparison using the NCBI ultrasonic atomizer, respectively. For liquid cultivation, BLAST database (Liu et al. 2012). The alga was maintained the alga inoculum was concentrated by gravity settling on solid BG11 medium containing 1 % agar and then trans- and was resuspended in fresh BBM medium to a cell ferred into liquid modified Bold’s Basal medium (BBM) for densityof0.5gL−1. culturing. The composition of the modified BBM medium All of the three types of bioreactor were installed in a was as follows (Abe et al. 2008): 1.0 g NH4Cl, 175 mg cultivation room under constant temperature (25 °C), contin- KH2PO4,75mgK2HPO4,25mgMgSO4·7H2O, 25 mg NaCl, uously aerated with 1 % CO2 gas at 0.1 vvm, and illuminated 50 mg EDTA, 30 mg KOH, 5 mg FeSO4·7H2O, and 11 mg with artificial light, provided by fluorescent lamps, of around −2 −1 H3BO3 in 1 L of deionized (DI) water (pH adjusted prior to 35 μmol photons m s . autoclaving to 8.0). The relative humidity in the glass chamber was deter- The strain was firstly grown in liquid BBM in a glass mined using a digital hygrometer (S-WS8061A, Shenzhen bubbling column (5-cm diameter and 700 cm high) under a AOV Testing Technology Co., Ltd., China). constant temperature of 25 °C and a light intensity of 50 μmol photons m−2 s−1. The culture was continuously Measurement of algae growth aerated with CO2-enriched air (air:CO2=1:0.02, v/v) at a flow rate of 0.25 vvm. After 7 days of cultivation, the broth was The chlorophyll content and dry weight were measured used as the inoculum. to evaluate algae growth. For the aquatic culture, 3–5mL of the culture medium was filtered on a pre-weighted NC Photo-bioreactor and cultivation membrane under vacuum and washed three times with deionized water. Then the membranes were oven dried to Three types of photo bioreactor were employed in this a constant weight at 105 °C for 2 h. For the aerial and study to cultivate T. arborum.Thefirst(Fig.1a)and subaerial cultures, the absolute amounts of the biomass second types (Fig. 1c) of bioreactor, which were called on the filter paper could not be determined because the “attached cultivation bioreactors” in our previous studies algal filaments could not be completely separated from (Cheng et al. 2013; Liu et al. 2013), consisted of the same the cellulose of the filter paper. Therefore, the pigments “single-layer plate” system with different medium- per sample were determined. The cells and the filter supplying modes. In the first type of reactor, the medium paper were sampled together and soaked in 5 mL J Appl Phycol

+ Microbial Sci. & Tech. Co., Ltd., China). The NH4 –Nin filter paper was washed off with 5 mL of hydrochloric acid −1 + solution (1 mol L ), and then the concentration of NH4 –N (CN) was determined. The amount of water in the same filter paper was calculated from the difference between the weight

before (Wo) and after (Wd) being dried at 105 °C for 2.5 h. The + −1 concentration of NH4 –N(gL ) in filter paper was calculated as follows: ÀÁ þ −1 NH4 –NgL ¼ CN Ã 5=ðÞW o−W d ð4Þ

Analyses of pigments

The pigments were extracted with DMSO, and absorbance was measured using a Varian 50 Bio UV-Visible spectropho- tometer (Varian Inc., USA). The chlorophyll and total carot- Fig. 1 The schematic diagrams of three photo-bioreactors. a Subaerial enoid contents were calculated as described by Wellburn cultivation. b Aquatic cultivation. c Aerial cultivation. Compressed air (1994): (contains 2 % CO2, v/v) is injected into the chambers or column ÀÁ −1 Chlorophyll a ðÞCa μgmL dimethyl sulfoxide (DMSO) and then stored in a cool ¼ : Ã ðÞ− : Ã ðÞ ðÞ anddarkconditionbeforeanalysis. 12 19 OD665 3 45 OD649 5 − The specific growth rate (μ,d 1) was calculated as ÀÁ −1 Chlorophyll b ðÞCb μgmL μ ¼ ðÞlnCt−lnC0 =t ð1Þ ¼ 21:99 Ã ðÞOD649 −5:32 Ã ðÞOD665 ð6Þ −1 where Ct and C0 represent the dry weight (g L ) or chloro- −2 ÀÁ phyll content (μgcm )atdayt and day 0, respectively, and t −1 Total carotenoidsðÞ C þ μgmL is the time of cultivation (days). x c

The volumetric productivity and areal productivity for total ¼ ðÞ1000 Ã OD480−2:14 Ã Ca−70:16 Ã Cb =220 ð7Þ carotenoid productivity were calculated as follows: ÀÁ −2 −1 = ¼ =ðÞþ ð Þ Productivity mg m day ¼ ðÞÃCt−Co 10=t ð2Þ Car Chl Total carotenoids Chlorophyll a Chlorophyll b 8 ÀÁ −1 −1 Productivity mg L day ¼ ðÞÃCt−Co =t ð3Þ Quantitative determination of carotenoids was carried out using HPLC according to Yuan et al. (2002). A liquid chro- −2 where the Ct and C0 are the total carotenoid content (μgcm matograph (Waters 1525, USA) equipped with a 996 photo- − or μgcm 3)atdayt and day 0, respectively, and t is the time of diode array detector (Waters 2998, USA) was used. The cultivation (days). pigments were separated on a Beckman Ultrasphere C18 reversed phase column (5 μm; 250×4.6 mm) at 30 °C. The Cell morphology observations mobile phase consisted of eluent A (dichloromethane: methanol:acetonitrile:water, 5.0:85.0:5.5:4.5, v/v)andel- Micrographs were taken with an Olympus SZX16 stereomi- uent B (dichloromethane:methanol:acetonitrile:water, croscope equipped with a DP72 digital camera and an Olym- 25.0:28.0:42.5:4.5, v/v). Separation of carotenoids was pus BX51 microscope equipped with a DP72 digital camera. achieved by the following gradient procedure: 0 % of B The cells were observed in dark field (DF) or bright field (BF) for 8 min, a linear gradient from 0 to 100 % of B within mode. 6 min, and 100 % of B for 40 min, at a flow rate of 1.0 mL min−1. The absorption spectra of carotenoids were Analysis of ammonium nitrogen displayed between 250 and 700 nm. Peaks were measured at 480 nm to facilitate the detection of carotenoids. The + The ammonium nitrogen (NH4 –N) was determined by using estimation of the quantity of the each carotenoid was an Ammonia Nitrogen Test Kit (Guangdong Huankai carried out from the chromatogram peak areas. J Appl Phycol

Fig. 2 Micrographs of Trentepohlia arborum grown under aquatic (a, b), subaerial (c– e), and aerial conditions (f–h), respectively, on day 12. All cultures were grown in full BBM, supplied with 2 % CO2.The irradiance was 35 μmol photons m2 s−1 and the temperature was 25 °C. All scale bars=200 μm, unless otherwise indicated

Statistical analysis in length and were poorly branched, the same as in the aquatic condition. A great number of orange droplets (likely lipid Statistical analysis was carried out using SPSS 11.0 software bodies) were observed in the cells from the aerial cultivation, (SPSS Inc., USA). ANOVAwas performed to evaluate signif- which made the filaments appear orange colored (Fig. 1h). On icance of individual differences with a probability threshold of the other hand, most droplets in the algae from the subaerial 0.05, followed by a post hoc Tukey test. cultivation were characterized by a green color and were smaller in size but copious in number (Fig. 2e). Interestingly, no sporangia and other reproductive structures were found at any time. Results Growth under varying culture conditions Morphological observations under three types of cultivation The growth rate of T. arborum under different conditions, The morphology of T. arborum cultivated in different habitats expressed as pigments per cubic centimeter or square centi- isshowninFig.2. In the aquatic condition, some algal meter culture, is compared in Fig. 3. All cultures were main- filaments were highly branched and aggregated into a tangled tained in the same BBM under a constant irradiance of −2 −1 mass in spherical colonies, which could grow up to about 35 μmol photons m s and temperature of 25 °C. The −1 500 μm in diameter (Fig. 2a). At the outer surface of the specific growth rates (μ,h ), as calculated in logarithmic spherical colonies, however, the algal filaments were poorly growth phase, were 0.007 (aerial), 0.034 (subaerial), and −1 branched and varied in length of tens to hundreds of micro- 0.010 h (aquatic). This clearly demonstrated that the algae meters (Fig. 2a, b). On the other hand, in the aerial and grew much faster in the subaerial condition compared to the subaerial conditions, T. arborum grew on the surface of the aquatic or aerial ones. filter paper to form an “algal carpet”, consisting of many compact colonies. However, the “carpet” exhibited a green Carotenoid accumulation in varying culture conditions color in the subaerial condition (Fig. 2c) and a yellow one in the aerial condition (Fig. 2f). When a section of the carpet was Figure 4 shows the ratio of total carotenoids to total chloro- observed microscopically, it was shown to consist of a top phylls (Car/Chl) in the algae grown in the three different layer of erect filaments and an intermediate layer of prostrate habitats. The total carotenoids and total chlorophylls in subaer- filaments (Fig. 2d, g). The erect filaments were up to 1684 μm ial and aquatic cultures rose progressively and proportionally J Appl Phycol

Fig. 3 The growth kinetics of Trentepohlia arborum grown under aquatic (a), subaerial (b), and aerial (c) conditions. All cultures were grown in full BBM, supplied with 2 % CO2,under 35 μmol photons m2 s−1 irradiance and 25 °C. Data and error bars represent means±SD of three replicates

during the whole experimental period (Fig. 3a, b) with the increased simultaneously. However, when the cells entered carotenoid to chlorophyll ratio (Car/Chl) remaining nearly into the nitrogen-deprived stage, although the content of total steady for the aquatic cultivation while slightly decreasing in pigments only had a slight increase, the total carotenoids the subaerial habitat (Fig. 4). In contrast, the total chlorophyll obviously increased, with a corresponding decline of chloro- content in the aerial culture was constant throughout the whole phylls, resulting in a dramatic increase in the Car/Chl ratio experimental period except for the first 3 days, while the total from 0.226 to 1.277 from day 12 to day 21. carotenoids increased strongly at the same time (Fig. 3c). Therefore, the Car/Chl ratio of the aerial habitat culture in- creased with cultivation time, reaching a maximum value of Discussion 0.99 after 12 days of cultivation (Fig. 4). The carotenoids and chlorophylls in the pigment extracts The microalga T. arborum presented different colonies and from T. arborum grown under different conditions were ana- filament shapes depending on the artificial habitat in which it lyzed using HPLC. The retention time (Rt) and visible spectra was grown. Typically, erect filaments were distributed on the peaks were used to identify carotenoids and chlorophylls, by top or outside of the colonies while the prostrate ones were comparing them with those of standards available in the laboratory and data from published papers. Zeaxanthin (Rt 9.3 min and visible spectra peaks at 445 and 473 nm) and β,β- carotene (Rt 54.9 min and visible spectra peaks at 454.6 and 481.3 nm), and chlorophyll b (Rt 24.6 min and visible spectra peaks at 464.3 and 647.5 nm) and chlorophyll a (Rt 30.0 min and visible spectra peaks at 431.5 and 664.7 nm) were found to be the main carotenoids and chlorophylls, respectively, in the pigment extracts from all three cultures (Table 1).

Enhanced carotenoid accumulation in subaerial cultivation by nitrogen starvation

In subaerial cultivation, T. arborum was grown initially in nitrogen-replete medium (full BBM) for 12 days and subse- quently in nitrogen-deprived medium by changing the bulk medium in the culture chamber. In this way, T. arborum Fig. 4 Ratio of total carotenoids to total chlorophylls (Car/Chl) in algae cultured in different habitats. All cultures were grown in full BBM, exhibited two stages of growth kinetics. As shown in Fig. 5, 2 −1 supplied with 2 % CO2,under35μmol photons m s irradiance and in the first nitrogen-replete stage, carotenoids and chlorophylls 25 °C. Data and error bars represent means±SD of three replicates J Appl Phycol

Table 1 Compositions of indi- vidual carotenoid in Trentepohlia Culture method Carotene profiles (% in total carotenoids) arborum under varying culture conditions after 12 days of Neoxanthin Lutein Zeaxanthin β,β-Carotene β,ε-Carotene cultivation Aquatic 2.5 1.2 33.1 49.9 13.2 Subaerial 2.0 1.7 62.9 27.8 5.6 Aerial 0.2 0.2 14.8 60.5 24.3

distributed at the bottom or inside of the colonies when grown observed for T. odorata on agar substrate by Lee et al. (1990). in aerial/subaerial or aquatic habitats (Fig. 2a, c, f). This Furthermore, vegetative survival of aerial algae might be phenomenon is probably due to the different direction of damaged by submersion. It has been shown that T. aurea illumination in the three culture habitats. Actually, in the started to disintegrate after a 1-month submersion (Gupta aquatic habitat, the light on the colony surface was checkered and Agrawal 2004), but how long-term submersion affects because of the agitation of bubbles, resulting in erect filaments T. arborum is not clear. On the other hand, variation of that grew omnidirectionally around the colonies. On the other medium supply affected the water conditions around algal hand, in aerial and subaerial habitats, the erect filaments grew cells, especially producing desiccation stress in aerial cultiva- unidirectionally, toward the light. In subaerial cultivation, tion with limiting water supply. Although the relative humid- some of the erect filaments began to detach from the aquatic ity was equal (95±3 % at 25.8 °C) for the algae grown under surface and formed aerial filaments (Fig. 2d, g). In preliminary aerial and subaerial conditions, the form and amount of water experiments, T. arborum had faster growth rate on cellulose available for algal cells were different. For subaerial cultiva- filter paper than on nitrocellulose and glass fiber ones, possi- tion, the prostrate filaments of T. arborum, i.e., the adhered bly due to differences in surface topography and better part of algal colonies, were completely soaked in a liquid bioaffinity (Irving and Allen 2011), which allowed the algal medium. On the other hand, in aerial cultivation, the algal filaments to adhere tightly by twining around the cellulose cells were surrounded by a mist of the medium, even if they fibers (Fig. 2d, g). In fact, the algal filaments were stable even were not directly in contact with the liquid medium. In the in the relative high flow rate of the medium, which means that present study, T. arborum exhibited a lower growth rate and the cells would not be easily washed off compared to other yellow-green filaments in aerial cultivation (Table 2 and algae (Cheng et al. 2013; Liu et al. 2013). The aerial filaments Fig. 2), which is an indicator of stress. The lower growth of are common for Trentepohlia in its natural habitat (Rindi and T. arborum might be caused by photosynthesis inhibition Guiry 2002; Rindi and Lopez-Bautista 2008), which probably induced by desiccation stress, which has been previously well allows the colonies to compete for moisture and light and documented for T. odorata (Ong et al. 1992), as well as for allows for the production of more biomass in a limited space. other aerial algae such as Nostoc flagelliforme (Ye et al. 2012), In the present study, the filaments of T. arborum grew in length up to 1684 μm in subaerial cultivation, while they grow up to 20 mm in natural habitats (Rindi et al. 2005). For attached or aquatic cultivation, the cells of T. arborum were in an immobilized or free state and were able to grow aerially/subaerially or aquatically. The microenvironment around filaments and cells varied with cultivation modes, affecting the growth. On the one hand, long-term submersion inhibited the reproduction of, especially sporangium forma- tion, when the alga was grown in the aquatic condition. This phenomenon was previously observed by Lee et al. (1990) and Gupta et al. (2004), who reported how T. odorata and T. aurea developed fewer or no sporangia under long-period submersion. We also confirmed that no sporangia developed in T. arborum in aquatic culture. Interestingly, T. arborum showed the ability to reproduce vegetatively from old or detached branches (Rindi et al. 2005), which might be the primary reproduction mode for this species. In subaerial cul- Fig. 5 Pigment yield in Trentepohlia arborum in both nitrogen-replete and nitrogen-deprived media in subaerial cultivation. The algae were tivation, the cellulose substrate might offer a solid support for grown at 25 °C and under 35 μmol photons m2 s−1 of irradiance. Data detached cells to attach to and form new colonies there, as and error bars represent means±SD of three replicates J Appl Phycol

Table 2 Growth of Trentepohlia spp. in various culture states

Species Specific growth State of culture State of cells Reference rate (μ,h−1)

Trentepohlia arborum 0.034 Subaerial Immobilized This study Trentepohlia arborum 0.007 Aerial Immobilized This study Trentepohlia arborum 0.010 Aquatic Free This study Trentepohlia aurea 0.005–0.032 Aquatic Free (Abe et al. 1998) Trentepohlia aurea 0.002a Aquatic Immobilized (Abe et al. 2003) Trentepohlia odorata 0.028 Aquatic Free (Tan et al. 1993) a Calculated from the reference

Bostrychia calliptera,andCaloglossa leprieurii (Pena et al. T. arborum (Fig. 5). In the present study, zeaxanthin was the 1999). primary carotene in subaerial cultivation while β,β-carotene T. arborum grew faster in the subaerial condition than in was the major carotenoid in aquatic and aerial cultivation the aquatic one, which is consistent with the performance of (Table 1), both of which have very high antioxidant activity Botryococcus braunii in a similar bioreactor (Cheng et al. and high commercial value (Abe et al. 1999; Mukherjee et al. 2013). Compared with other species in the same genus, the 2010). specific growth rate (μ)ofT. arborum was much higher than In the present work, T. arborum showed a relatively high that of T. aurea when grown aquatically and as immobilized carotenoid productivity of 0.69 mg L−1 day−1, which was cells but was slightly lower than that of T. aurea and obtained in aquatic cultivation, and was much higher than that T. odorata, for free cells (Tan et al. 1993; Abe et al. 1998). of 0.17 mg L−1 day−1 in T. aurea (Abe et al. 1999). As for Therefore, T. arborum exhibited the fastest growth perfor- attached cultivation, the carotenoid productivity was 4.88 and mance for immobilized cells among studied species, and it is 15.99 mg m−2 day−1 in aerial and in subaerial conditions, a promising species for attached cultivation. respectively. Although no attempt was made to enhance ca- The pigment profiles in T. arborum varied among the rotenoid production in this study, some methods which could different cultivation methods. The Car/Chl ratio was steady be used to achieve this objective, such as nutrient starvation during the whole experiment period, except for a successive (Tan et al. 1993; Abe et al. 1998), desiccation stress (Ong et al. increment in aerial cultivation (Fig. 4).Thelikelymainreason 1992), or high light (Tan et al. 1993; Ortega-Morales et al. was the long-term desiccation stress in the aerial condition, in 2013), have been investigated. Interestingly, the algae cells which water supply might be relatively insufficient compared and bulk medium were separated in attached cultivation as to that of the other two bioreactors as stated above. A similar mentioned above, as well as in our previous reports (Ji et al. result was reported by Ong and Lim (1992) who found that the 2014; Liu et al. 2013). Therefore, the transition to nutrient- Car/Chl ratio could increase to 25:1 under more severe and deprived mode from nutrient-replete one can be done easily longer desiccation. Moreover, nitrogen starvation, in spite of a and swiftly by exchanging the bulk medium without any sufficient humidity supply, also increased the Car/Chl ratio in treatment of the algae, which is an additional benefit of this

Table 3 Carotenoid productivity of Trentepohlia spp. and Species Carotenoid productivity State of culture Reference Dunaliella salina Unit (mg L−1 day−1) Trentepohlia arborum 0.69 Aquatic This study Trentepohlia aurea 0.17 Aquatic (Abe et al. 1999) Unit (mg m−2 day−1) Trentepohlia arborum 4.88 Aerial This study a Algae grown in nitrogen-replete Trentepohlia arborum 15.99a Subaerial This study culture in attached cultivation Trentepohlia arborum 67.7b Subaerial This study b Algae grown in nitrogen- Dunaliella salina 13.5 Aquatic (Kleinegris et al. 2011) deprived culture in attached culti- vation. Productivity value was Dunaliella salina 40–90 Aquatic (Garcia-Gonzalez et al. calculated based on time required 2003) by the nitrogen-deprived stage of Dunaliella salina 200 Aquatic (Borowitzka 2013b) cultivation J Appl Phycol culture mode. The carotenoid production was successfully and immunofluorescence cytochemistry. Int J Syst Evol Microbiol – accelerated by changing the nitrogen-replete medium in the 51:759 765 Cheng PF, Ji B, Gao LL, Zhang W, Wang JF, Liu TZ (2013) The growth, culture chamber to a nitrogen-deprived medium, resulting in a lipid and hydrocarbon production of Botryococcus braunii with − − carotenoid productivity of 67.7 mg m 2 day 1, which is almost attached cultivation. Bioresour Technol 138:95–100 fourfold of that achieved under the nitrogen-replete condition. Czeczuga B, Maximov OM (1996) Carotenoids in the cells of the alga – This productivity, obtained under our experimental condi- Trentepohlia gobii Meyer. Acta Soc Bot Pol 65:273 276 Garcia-Gonzalez M, Moreno J, Canavate JP, Anguis V, Prieto A, tions, is lower than the best value reported for the alga Manzano C, Florencio FJ, Guerrero MG (2003) Conditions for Dunaliella salina (Table 3), but it was much higher or com- open-air outdoor culture of Dunaliella salina in southern Spain. J parable to those reported by Kleinegris et al. (2011)and Appl Phycol 15:177–184 Garcia-Gonzalez et al. (2003) for the same strain. It should Gupta S, Agrawal SC (2004) Vegetative survival and reproduction under β submerged and air-exposed conditions and vegetative survival as be pointed out that D. salina is the most promising alga for - affected by salts, pesticides, and metals in aerial green alga carotene production and it has been extensively cultivated on a Trentepohlia aurea. Folia Microbiol 49:37–40 commercial scale for years (Jin and Melis 2003;Borowitzka Ho KK, Tan KH, Wee YC (1983) Growth-conditions of 2013a), while no previous reports are available in the literature Trentepohlia odorata (Chlorophyta, Ulotrichales). Phycologia 22:303–308 for T. arborum. Irving TE, Allen DG (2011) Species and material considerations in the In summary, the growth and carotenoid accumulation of formation and development of microalgal biofilms. 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