Outdoor Mass Culture Research Articles

Aerial microalgae Coccomyxa simplex isolated from a low-temperature, low-light environment, and its biofilm growth and lipid accumulation

Biofuels produced from renewable resources could be an effective and sustainable alternative to fossil fuels. However, biodiesel production from microalgae is slow during cold periods, which are unfavorable for the growth of conventional microalgae, especially in outdoor mass cultivation. We screened and isolated aerial microalgae that are able to grow at lower temperatures and light intensity than conventional algae. Seven aerial microalgae were isolated from the surface of rocks in a mountainous district in Japan. These microalgae were identified as Coccomyxa simplex (KGU-H002–008 strains), class Trebouxiophyceae, based on 18S rRNA gene analysis. These algal strains were cultured at 15 °C and 15 μmol photons m−2 s−1 until nitrogen was depleted in the liquid culture medium. Their biomass reached 0.7–1.5 g (dry cell weight) per L culture medium. The fatty acid contents of KGU-H002, -H004, and -H005 were approximately 150 mg g−1 dry cell weight (dcw), and these strains produced abundant C16 and C18 fatty acids. We prepared biofilms of C. simplex KGU-H002, -H004, and -H005, and examined their biomass and fatty acid accumulation in the aerial phase at 15 or 25 °C under low light intensity. Under both temperature conditions, the three strains showed a similar increase (~2.2-fold) in biomass. In KGU-H002 and -H004, the fatty acid content was higher at 15 °C than at 25 °C. These results indicate that aerial cultures of C. simplex KGU-H002 and -H004 have potential applications in biodiesel production under low temperature and low light intensity.

Light supply and mineral nutrition conditions as optimization factors for outdoor mass culture of carotenogenic microalga Dunaliella salina

The productivity characteristics of green halophilic algae Dunaliella salina during its pilot cultivation were compared between two nutrient media and under different illumination. Cultivation was carried out in ponds located in the greenhouse during summer season. Two main factors that determine rates of growth and pigments synthesis, as well as the pigments ratio in D. salina grown outdoors in summer, are light and temperature conditions. The mean productivity of D. salina culture grown in two nutrient media, differing in nutrients concentration, was the same, and the mean carotenoid accumulation rate was 30% higher when using Ben-Amotz medium (52 mg·m−2·day−1) than Johnson medium. D. salina cell illumination was decreased by pond shading or by doubling their depth. Both these methods allowed reducing the level of the stress impact on algal culture under extreme light and temperature conditions. Shading of the ponds resulted in an increase in biomass and carotenoid productivity by 30% and 50%. However, a two-fold increase in ponds depth proved to be a more effective technological method, which allowed for an increase in D. salina areal productivity both in biomass and carotenoids by a factor of 3.5–3.8. In conclusion, less concentrated medium and increasing culture depth may be recommended as the optimization factors for D. salina outdoor cultivation.

Chlororespiration protects the photosynthetic apparatus against photoinhibition by alleviating inhibition of photodamaged-PSII repair in Haematococcus pluvialis at the green motile stage

During outdoor mass cultivation, Haematococcus pluvialis cells, especially at the green motile stage, are frequently exposed to high light. However, the photoprotective mechanisms in H. pluvialis have not been well characterized. This study aimed to confirm the photoprotection of chlororespiration in H. pluvialis at the green motile stage. Under high light, inhibition of plastid terminal oxidase (PTOX), a key enzyme of chlororespiration, increased the relative variable fluorescence at the I-step (VI), causing a decrease in the plastoquinone (PQ) pool, which further led to photosystem II (PSII) acceptor side over-reduction. This condition disturbed the balance between light absorption and energy utilization, causing the over-excitation of PSII reaction centers, which was indicated by the fact that the inhibition of PTOX increased the relative variable fluorescence at the J-step (VJ) and the light absorption flux per active reaction center (ABS/RC), but decreased the electron transport flux per active reaction center (ETo/RC) in H. pluvialis. The over-reduction of the PSII acceptor side and the over-excitation of PSII reaction centers inevitably caused more severe photoinhibition as shown by a decrease in Fv/Fm (maximal photochemical efficiency of PSII). Furthermore, the level of photoinhibition was not changed when the PTOX was inhibited in the presence of the photodamaged-PSII repair inhibitor chloramphenicol, indicating that the inhibition of PTOX did not accelerate the photodamage to PSII directly. The above results suggest that the chlororespiration pathway protects H. pluvialis at the green motile stage against photoinhibition by decreasing the inhibition of the photodamaged-PSII repair mechanism.

Trial of stable outdoor mass culture of Chaetoceros calcitrans

Trial of stable outdoor mass culture of <I>Chaetoceros calcitrans</I>

Growth, biomass and lipid productivity of a newly isolated tropical marine diatom, Skeletonema sp. UHO29, under different light intensities

Abstract. Indrayani I, Haslianti H, Asmariani A, Muskita WH, Balubi M. 2020. Growth, biomass and lipid productivity of a newly isolated tropical marine diatom, Skeletonema sp. UHO29, under different light intensities. Biodiversitas 21: 1498-1503. Light is one of the important factors affecting growth and biochemical composition of microalgae. The aim of this study was to determine growth, biomass and lipid productivity of a newly isolated marine diatom, Skeletonema sp.UHO29 under different light intensities. The Skeletonema sp. was cultured in 300 mL conical flasks containing 150 mL of f/2 medium under three different light intensities (28, 60 and 110 μmol photons m-2 s-1 ) with three replications. The cultures were incubated under ambient room temperatures, and a cycle of 12 hours light and 12 hours dark, in batch modes for two weeks. The results showed that the highest specific growth rate (0.56 d-1) was achieved at the highest light intensity (110 μmol photons m-2 s-1) and the lowest (0.11 d-1) was obtained at the lowest light intensity (28 μmol photons m-2 s-1). The highest biomass yield (0.62 g L-1) and biomass productivity (0.34 g L-1 d-1) was achieved at the highest light intensity. However, the highest lipid yield (0.175 g L-1), lipid content (28.78% ash-free dry weight) and lipid productivity (0.067 g L-1 d-1) were achieved at medium light intensity. The alga prefers high light intensity for higher growth rate and biomass productivity but it produces more lipids at medium light intensity. This study indicated that the Skeletonema sp.UHO29 is a potential species for outdoor mass cultivation and is a promising microalgal strain for use as a biodiesel feedstock due to its high growth rate, high biomass and lipid productivity.

The hormesis effects of low-dose 60Co gamma irradiation on high-temperature tolerance in cultivated Sargassum horneri (Fucales, Phaeophyceae)

Radiation hormesis is a phenomenon in which low doses of ionizing radiation can have stimulatory effects on biological systems. The temperate brown alga Sargassum horneri was the subject of this study on the hormetic effects of low-dose 60Co gamma radiation on macroalgae incubated in vitro at high temperatures. Sargassum horneri embryos were irradiated with 15, 20, and 25 Gy 60Co radiation and then incubated in vitro at 18, 21, 24, 27, and 30 °C. The same radiation treatment was also applied to S. horneri embryos grown in situ in outdoor mass cultivation. Non-irradiated in vitro control cultures had optimal growth at 21 °C, but irradiated cultures had optimal growth at 27 °C and growth was significantly enhanced by 32% compared to the control cultures at 21 °C. In contrast, in situ cultures of S. horneri in outdoor 10-ton seawater tanks showed that irradiated thalli had less total biomass as compared to non-irradiated control thalli. While low doses of gamma radiation may induce high-temperature tolerance resulting in better growth rates under the highly controlled in vitro culture conditions, these effects were not clearly observed in the in situ outdoor mass-cultivation.

Tetraselmis suecica F&M-M33 growth is influenced by its associated bacteria.

SummaryAlgal cultures are usually co‐cultures of algae and bacteria, especially when considering outdoor mass cultivation. The influence of associated bacteria on algal culture performance has been poorly investigated, although bacteria may strongly affect biomass (or derived product) yield and quality. In this work, the influence on growth and productivity of Tetraselmis suecica F&M‐M33 of bacterial communities and single bacterial isolates from the algal phycosphere was investigated. Xenic laboratory and outdoor cultures were compared with an axenic culture in batch. The presence of the bacterial community significantly promoted culture growth. Single bacterial isolates previously found to be strictly associated with T. suecica F&M‐M33 also increased growth compared with the axenic culture, whereas loosely associated and common seawater bacteria induced variable growth responses, from positive to detrimental. The increased growth was mainly evidenced as increased algal biomass production and cell size, and occurred after exhaustion of nutrients. This finding is of interest for biofuel production from microalgae, often attained through nutrient starvation processes leading to oil or carbohydrate accumulation. As axenic T. suecica F&M‐M33 showed a similar growth with or without vitamins, the most probable mechanism behind bacterial positive influence on algal growth seems nutrient recycling.

Development of large-scale and economic pH control system for outdoor cultivation of microalgae Haematococcus pluvialis using industrial flue gas

The aim of this study was to develop the economic and effective buffer system for microalgae mass cultivation using industrial flue gas. Due to the continuous flue gas supplement, culture media acidified, therefore cell growth inhibited. Although buffering agent was added, this result increase in cost for overall culture process. Therefore combined buffer system of bicarbonate and phosphate (BP) for large-scale use was investigated. The bicarbonate buffer system generated from CO2 dissolution, additionally phosphate buffer system improves the buffer performance under the continuous CO2 supplementation from flue gas. The microalgae Haematococcus pluvialis was cultivated under autotrophic outdoor conditions using these buffer solutions. As a result, the autotrophic BP buffer system enhanced the biomass and astaxanthin productivity of H. pluvialis to 105% and 103%, respectively. The results confirm that the BP buffer system reduces the cost of microalgal CO2 conversion process, particularly for the outdoor mass cultivation.

Outdoor Cultivation of Marine Diatoms for Year-Round Production of Biofuels.

Biofuel production using microalgae is believed to have the advantage of continuous year-round production over crop plants, which have strong seasonality. However, actual year-round production of microalgal lipids using outdoor mass cultivation has rarely been demonstrated. In our previous study, it was demonstrated that the oleaginous diatom, Fistulifera solaris, was culturable in outdoor bioreactors from spring to autumn, whereas biomass and lipid production in winter failed because F. solaris did not grow below 15 °C. Therefore, another candidate strain that is culturable in winter is required. In this study, a cold-tolerant diatom, Mayamaea sp. JPCC CTDA0820, was selected as a promising candidate for biofuel production in winter. Laboratory-scale characterization revealed that this diatom was culturable at temperatures as low as 10 °C. Subsequently, F. solaris (April–October) and Mayamaea sp. JPCC CTDA0820 (November–March) were cultured in outdoor open-pond bioreactors, wherein year-round production of diatom lipids was successfully demonstrated. The maximal values of areal productivities of biomass and lipids reached to 9.79 and 1.80 g/(m2 day) for F. solaris, and 8.62 and 0.92 g/(m2 day) for Mayamaea sp. JPCC CTDA0820, respectively. With the combined use of these two diatom species, stable year-round production of microalgal lipids became possible.

Large-Scale Cultivation of Euglena.

From the middle of the twentieth century, microalgae have been exploited as a candidate biomass source of food and other products. One such candidate source is the fast-proliferating microalga Euglena gracilis. The commercial cultivation of E. gracilis began in 2007, after the success of its outdoor mass cultivation and improvement of the harvesting and drying methods suitable for Euglena cells. The commercialization of Euglena production is based on the strategy of "5Fs of Biomass," which refers to the development and production of commercial products including food, fiber, feed, fertilizer, and fuel from biomass." Although room for improvement remains in the productivity of Euglena biomass, the product with the highest value-food-is already profitable. By enhancing the productivity of its biomass, other Euglena products, including fiber, feed, fertilizer, and fuel, can be commercialized. Breeding and recombinant DNA technology studies are being conducted to accomplish more extensive application of Euglena. In addition, the search for a better place for outdoor mass cultivation of Euglena is ongoing.

Use of commercial probiotics for the improvement of water quality and rotifer density in outdoor mass culture tanks

An experimental study was carried out to evaluate the efficacy of three commercially available probiotics P1, P2 and P3 consisting of mainly Bacillus spp. and nitrifying bacteria against Vibrio loads in mass culture tanks of the rotifer Brachionus plicatilis. Triplicate tanks were maintained for each of the probiotic treatment as well as for control group. All the tanks were inoculated with 50 rotifers ml-1 and were fed with Nannochloropsis oculata at a density of 1 x 107 cells ml-1. Every alternate day, all the experimental tanks were treated with probiotics at a concentration of 1 x 104 cfu ml-1 and the experiment was carried out for one week. The study showed a significant increase in rotifer density (p&lt;0.05) in all the tanks treated with the probiotics and a maximum density of 400 nos. ml-1 was observed in the tanks treated with P3. After 5th and 6th day of culture, total elimination of Vibrios was also recorded in the tanks treated with P3 and P2 respectively. The study revealed that P3, with a combination of Bacillus, Thiobacillus, Acetobacter and Paracoccus supplemented with enzymes, was found to be most effective in the enhancement of rotifer density and also in the elimination of Vibrios in rotifer mass culture tanks.

An enclosed rotating floating photobioreactor (RFP) powered by flowing water for mass cultivation of photosynthetic microalgae.

BackgroundThe design of photobioreactor (PBR) for outdoor mass cultivation of microalgae determines the distribution of solar irradiance among cells in the culture, mode of agitation, mass transfer efficacy, and energy consumption, thus determines the productivity of the system and the cost of production. In this study, the concept of a floating photobioreactor with rotation function is proposed. Dunaliella tertiolecta, a model microalga, cultured in the attached vessels was evaluated.ResultsThe rotation of the photobioreactor was powered by flowing water, in this case waves generated through a paddle wheel in an outdoor raceway pond for proof of concept. The rotating floating PBR (RFP) could be powered by natural flowing stream, river, and tidal waves, thus there could be no energy cost for agitation of the cultures in maintaining the cells in suspension. This RFP is characterized by its energy-saving and temperature control properties as well as more homogenous light distribution in the culture as compared to conventional culture systems, such as raceway pond. Maximal cell concentration of 8.38 × 106 cells mL−1, biomass productivity of 3.10 g m−2 day−1, and photosynthetic efficiency of 4.61 % (PAR) were achieved. In addition, satisfactory productivities of D. tertiolecta metabolites including carotenoids, mycosporine-like amino acids and lipids were also obtained.ConclusionsThe RFP, powered by flowing water, creates an innovative culture technology for economical cultivation of microalgal cells and production of microalgal metabolites.

The bacterial community associated with Tetraselmis suecica outdoor mass cultures

Interactions between algae and associated bacterial communities in the phycosphere depend on many factors such as culture age, nutrient availability, and antibiotic production and greatly influence algal survival and growth. The microbial community associated with the marine microalga Tetraselmis suecica F&M-M33 in a laboratory culture and an outdoor mass culture, set up from the laboratory one, run during a whole year was investigated in different seasons through isolation in pure culture of bacteria, amplified ribosomal DNA restriction analysis (ARDRA) of all the isolates, and terminal restriction fragment length polymorphism (T-RFLP) analysis. The total number of bacterial isolates was 152, which clustered in thirty-six 16S rDNA groups by ARDRA. Sequencing of a representative of each ARDRA group permitted identification of bacterial genera never reported before in association with microalgae in outdoor mass cultures, although most of them were previously found to be associated with the marine environment (e.g., seawater, sea sediments). T-RFLP analysis revealed that spring and autumn bacterial community profiles were closely related, while the bacterial laboratory community was considerably different (less than 50 % similarity) from that associated with the outdoor culture in different seasons. T-RFLP results suggest the presence of a core of bacteria that are closely associated with the alga and of a part of the community which varies seasonally.

Modeling the competition between antenna size mutant and wild type microalgae in outdoor mass culture

Under high light conditions, microalgae are oversaturated with light which significantly reduces the light use efficiency. Microalgae with a reduced pigment content, antenna size mutants, have been proposed as a potential solution to increase the light use efficiency. The goal of this study was to investigate the competition between antenna size mutants and wild type microalgae in mass cultures. Using a kinetic model and literature-derived experimental data from wild type Chlorella sorokiniana, the productivity and competition of wild type cells and antenna size mutants were simulated. Cultivation was simulated in an outdoor microalgal raceway pond production system which was assumed to be limited by light only. Light conditions were based on a Mediterranean location (Tunisia) and a more temperate location (the Netherlands). Several wild type contamination levels were simulated in each mutant culture separately to predict the effect on the productivity over the cultivation time of a hypothetical summer season of 100days. The simulations demonstrate a good potential of antenna size reduction to increase the biomass productivity of microalgal cultures. However, it was also found that after a contamination with wild type cells the mutant cultures will be rapidly overgrown resulting in productivity loss.

Comparison of heat resistance and application potential of two lipid-rich Isochrysis galbana strains

Isochrysis galbana is considered to be a promising candidate for biodiesel feedstock for its fast growth and high lipid content. Outdoor mass cultivation of this species often suffers from heat stress, especially at noon in summer, thus screening of heat-resistant strains is essential. The heat resistance and application potential of two I. galbana strains IOAC683S and IOAC724S were compared for their growth characteristics, Hsp90 gene expression, photoprotective mechanism, fatty acid profile and lipid productivity at 25°C, 30°C and 35°C. Cell density, total chlorophyll and biomass of strain IOAC683S increased at 25°C and 30°C, while decreased at 35°C. In contrast, all these indexes of strain IOAC724S increased gradually in all three temperatures during 144h culture period. The HSP90 mRNA expression levels of strain IOAC683S increased significantly after cultured at 35°C for 24h (P<0.05), but that of strain IOAC724S did not increase significantly at 72h at 35°C compared with 25°C (P>0.05). These results indicated that strain IOAC724S was more resistant to 35°C than strain IOAC683S. It was found that, the lower chlorophyll content per cell in IOAC724S could reduce the light energy absorption, and higher non-photochemical energy quenching (NPQ) and alternative electron flow with PSII (AEF-II) could efficiently dissipate excess light energy, therefore protect it against photodamage at high temperature of 35°C. In addition, strain IOAC724S has higher lipid productivity than strain IOAC683S, giving this unicellular alga potential for outdoor commercial cultivation for biofuel production.

Mass Cultivation of Indian Microalgae for Bio-Fuel Production Under Outdoor Conditions of Visakhapatnam India East Coast

Background: Microalgae have been identified as potential biodiesel feedstock because of their high lipid and biomass productivity. The present work was aimed to the mass culture of microalgae in a shallow pond and circular tank under open-air condition and to determine the productivity of biomass, lipid, and fatty acid methyl esters. Methods: Chlorella vulgaris (GenBank: KF937218.1) isolated from the offshore water of Visakhapatnam along the East coast of India and Platymonas convolutae obtained from CMFRI, India, were mass cultured in a shallow pond and circular tank under open-air conditions. Outdoor mass cultivation was carried out for both the species in shallow pond and circular tank. The daily productivity and growth kinetics were calculated. Results: C. vulgaris had the highest biomass concentration (0.98 g l-1) with 21% of the lipid content in the circular tank culture. The maximum aerial productivities of 42 g m2 d-1 and 32.1 g m2 d-1 were recorded for C. vulgaris and P. convolutae, respectively. Fatty acid profiles showed the presence of C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, and C18:3. FAME yield determined via different extraction methods was also reported. Conclusion: The results suggest that the coastal area of Visakhapatnam is a feasible location for the production of biomass via large-scale mass culture in an open-air shallow pond. Fatty acid profile showed the feasibility production of biodiesel. Keywords: Biomass, Fatty acids, Open-air pond, growth kinetics, FAME, Chlorella vulgaris, Platymonas convolutae.

Bacterial Flora of Water and Rotifers in Outdoor Mass Culture Tanks Fed with Different Microalagal Diets

Investigations were carried out on the bacterial flora of water and the quality of rotifers in outdoor mass culture tanks. Brachionus plicatilis and B. rotundiformis were cultured in 10 numbers of 1 tonne tanks and the physico-chemical parameters and microbial loads of water, and total bacterial and Vibrio loads of microalgae and rotifers, during the period 2014-15, were studied. The study revealed a significant variation of the ammonia levels and total bacterial loads with different diets (P < 0.05). A positive correlation was observed between the total bacterial loads of algal concentrations and loads of rotifers, but no correlation was observed between Vibrio loads of water and rotifers. The prevalence of Vibrio loads of rotifers was low in tanks fed with Nannochloropsis oculata and maximum Vibrio loads were recorded in Chaetoceros calcitrans fed rotifers. The Vibrio loads significantly varied with days of culture and also with the algal diets. A positive correlation was observed between the total Vibrio loads of water and rotifers.

Growth and lipid content at low temperature of Arctic alga Chlamydomonas sp. KNM0029C.

Biodiesel produced from microalgae is a promising source of alternative energy. In winter, however, outdoor mass cultivation for biodiesel production is hampered by poor growth. Here, we report that Arctic Chlamydomonas sp. KNM0029C exhibits optimal growth at 4 °C and reaches densities up to 1.4 × 10(7) cells mL(-1). Lipid body formation in the alga was visualized through BODIPY 505/515 staining and fluorescence microscopy. The fatty acid methyl ester (FAME) production level of KNM0029C was 178.6 mg L(-1) culture and 2.3-fold higher than that of C. reinhardtii CC-125 at 4 °C. Analysis of the FAME content showed a predominance of polyunsaturated fatty acids such as C16:3, C18:2, C18:3, and C20:2. C18:3 fatty acids comprised the largest fraction (20.7%), and the content of polyunsaturated fatty acids (39.6%) was higher than that of saturated fatty acids (6.8%) at 4 °C. These results indicate that Chlamydomonas sp. KNM0029C, as a psychrophilic microalga, might represent a favorable source for biodiesel production in cold environments.

Growth model for raceway pond cultivation of Desmodesmus sp. MCC34 isolated from a local water body

Biofuel production by microalgae has the advantage of higher biomass productivity over land crops. The selection of potential microalgae depends on the growth in outdoor mass cultivation during different seasons, which can be predicted by a mathematical model. Here, freshwater green algae were isolated from a local water body in Pilani, Rajasthan, India (geographical coordinates: 28°22′N 75°36′E) and characterized by microscopy and ribosomal RNA analysis. The strain was submitted to the Indian Agricultural Research Institute's microbial culture collection (IARI, India) and identified as Desmodesmus sp. MCC34. This strain, along with a fresh water green algae (Chlorella minutissima), two marine green algae species (Dunaliella salina and Dunaliella tertiolecta) and two nitrogen fixing cyanobacteria (Nostoc muscorum and Anabaena doliolum), were screened for lipid productivity and growth kinetics under culture room and raceway pond conditions. Desmodesmus sp. MCC34 showed the highest specific growth rate (0.26 day−1), biomass production (1.9 g L−1) and lipid productivity (103 mg L−1 day−1). The optimal temperature and saturating light intensity for maximal growth of Desmodesmus sp. MCC34 were 35 °C and 75 μmol m−2 s−1 with molar extinction coefficient of 0.22 m2 g−1, respectively. Desmodesmus sp. MCC34 was then subjected to outdoor cultivation in a 20‐m long raceway pond for 18 days during March and November 2013. The areal biomass productivity and volumetric biomass productivity were 13946.23 kg ha−1 year−1 and 56.94 mg L− 1day−1 during the month of March, decreasing to 6262.28 kg ha−1 year−1 and 25.57 mg L− 1day−1 during the month of November. A mathematical model was constructed to explain the relationship between biomass production and growth parameters such as temperature, light intensity and nutrient concentration. The productivity values predicted with the proposed model correspond well with the experimental data, suggesting the validity of the model.

Photosynthesis of Scenedesmus obliquus in outdoor open thin-layer cascade system in high and low CO2 in Belgium

Two outdoor open thin-layer cascade systems operated as batch cultures with the alga Scenedesmus obliquus were used to compare the productivity and photosynthetic acclimations in control and CO2 supplemented cultures in relation with the outdoor light irradiance. We found that the culture productivity was limited by CO2 availability. In the CO2 supplemented culture, we obtained a productivity of up to 24gdwm−2day−1 and found a photosynthetic efficiency (value based on the PAR solar radiation energy) of up to 5%. In the CO2 limited culture, we obtained a productivity of up to 10gdwm−2day−1 while the photosynthetic efficiency was up to 3.3% and decreased to 2.1% when the integrated daily PAR increased. Fluorescence and oxygen evolution measurements showed that ETR and oxygen evolution light saturation curves, as well as light-dependent O2 uptake were similar in algal samples from both cultures when the CO2 limitation was removed. In contrast, we found that CO2 limitation conducted to a decreased PSII photochemical efficiency and an increased light-induced heat-dissipation in the control culture compared to the CO2 supplemented culture. These features are in line with a lower light use efficiency and may therefore contribute to the lower productivity observed in absence of CO2 supplementation in outdoor mass cultures of S. obliquus.