Some strains from microalgae collection IBASU-A (Ukraine) as an object of biotechnology

An information on the collection of strains of biotechnological application as an integral part of Microalgal Culture Collection of the M.G. Kholodny Institute of Botany of NAS of Ukraine (IBASU-A) is given. The base of its funds contains some green algal strains belonging to the families of Dunaliellaceae, Chlorellaceae, Scenedesmaceae and Selenastraceae. They have been isolated from different regions of Ukraine in order to find cultures of phototrophic microorganisms – promising for biotechnology, in particular, obtaining biologically active additives for the needs of the food industry, medicine, agriculture, raw materials for the production of biofuels, as well as bioindication, biomonitoring, bioremediation of aquatic objects of the environment, etc. Overall, this special collection includs 90 strains of halophile and freshwater microalgae of 30 species, 15 genera, 7 families, 4 orders, 2 classes. All of them are considered as important objects for industrial cultivation, solution of environmental problems, and the basis for further biotechnological research.

Assessing the Viability of Commercial Media for the Mass Culture of Chaetoceros muelleri

The microalgae Chaetoceros muelleri is considered a highly nutritious feed for the cultured larvae of the tropical sea cucumber Holothuria scabra. Due to the cost of analytical grade culture media used in the production of C. muelleri, there is a need to evaluate cheap alternative commercial media to decrease the cost of producing quality live microalgal food. In this study, two different indoor batch culture systems (1 L glass bottles and 10 L plastic carboys) were used to evaluate the effectiveness of two conventional (modified F/2 and Walne’s) and one commercial (Epizyme AGP complete) microalgal culture media. Results of the 1 L glass bottle experiment showed that the peak cell density of C. muelleri in AGP (1,241 ± 116 x 104 cells ml-1) was not significantly different from the modified F/2 (1,584 ± 41 x 104 cells ml-1) and Walne’s medium (1,319 ± 162 x 104 cells ml-1) (Kruskal-Wallis test, p=0.78). Likewise, in the plastic carboy experiment, the maximum cell density of C. muelleri in Walne’s medium (750 ± 144 x 104 cells ml-1) and F/2 medium (653 ± 79 x 104 cells ml-1) were higher, but not significantly different from AGP (496 ± 184 x 104 cells ml-1) (Kruskal-Wallis test, p=0.43). The highest growth rate in the glass bottle cultures was the modified F/2 (0.38 div day-1), while AGP was the lowest (0.34 div. day-1). On the other hand, in carboy culture, AGP was higher (0.17 div.day-1) compared to modified F/2 (0.15 div. day-1) and Walne’s medium (0.13 div. day-1). The exponential growth phase was similar in the glass bottles, while in the carboy, the exponential phase was reached at a shorter time in the AGP treatment than those in the modified F/2 and Walne’s media. The findings showed that AGP medium is an adequate alternative to replace the conventional media (modified F/2 and Walne’s) during the secondary stock culture for C. muelleri. The viability of using cheaper and more readily available commercial AGP media for the indoor culture production of C. muelleri can contribute to cost-effective scaling-up of the hatchery production of quality H. scabra larvae and early juveniles.

Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

Enhancement of co-production of lutein and protein in Chlorella sorokiniana FZU60 using different bioprocess operation strategies

Co-production of multiple compounds is an efficient approach to enhance the economic feasibility of microalgae-based metabolites production. In this study, Chlorella sorokiniana FZU60 was cultivated under different bioprocess strategies to enhance the co-production of lutein and protein. Results showed that both lutein and protein content (7.72 and 538.06 mg/g, respectively) were highest at the onset of nitrogen deficiency under batch cultivation. Semi-batch III strategy, with 75% microalgal culture replacement by fresh medium, obtained similar content, productivity, and yield of lutein and protein as batch cultivation, demonstrating that it can be used for stable and continuous production. Fed-batch II strategy, feeding with 1/3 modified BG11 medium, achieved super-high lutein and protein yield (28.81 and 1592.77 mg/L, respectively), thus can be used for high-output production. Besides, two-stage strategy, combining light intensity shift and semi-batch cultivation, gained extremely high lutein and protein productivity (15.31 and 1080.41 mg/L/day, respectively), thereby is a good option for high-efficiency production. Moreover, the fed-batch II and two-stage strategy achieved high-quality lutein and protein, thus are promising for the co-production of lutein and protein in C. sorokiniana FZU60 for commercial application.

The Effects of Total Dissolved Carbon Dioxide on the Growth Rate, Biochemical Composition, and Biomass Productivity of Nonaxenic Microalgal Polyculture

The biosequestration of CO2 using microalgae has emerged as a promising means of recycling CO2 into biomass via photosynthesis, which could be used to produce biofuels as an attractive approach to CO2 mitigation. We investigated the CO2 fixation capability of the native nonaxenic microalgal culture using a 2 L photobioreactor operated in batch mode. The cultivation was carried out at varying concentrations of total dissolved CO2 (Tco2) in the bulk media ranging from 200 to 1000 mg L−1, and the temperature and light intensities were kept constant. A maximum CO2 fixation rate was observed at 400 mg L−1 of Tco2. Characteristic growth parameters such as biomass productivity, specific growth rate, maximum biomass yield, and biochemical parameters such as carbohydrate, protein, and lipids were determined and discussed. We observed that the effect of CO2 concentration on growth and biochemical composition was quite significant. The maximum biomass productivity was 22.10 ± 0.70 mg L−1 day−1, and the rate of CO2 fixation was 28.85 ± 3.00 mg L−1 day−1 at 400 mg L−1 of Tco2. The maximum carbohydrate (8.17 ± 0.49% dry cell weight) and protein (30.41 ± 0.65%) contents were observed at 400 mg L−1, whereas the lipid content (56.00 ± 0.82% dry cell weight) was the maximum at 800 mg L−1 of Tco2 in the bulk medium.

Chromochloris zofingiensis (Chlorophyceae) Divides by Consecutive Multiple Fission Cell-Cycle under Batch and Continuous Cultivation

Several green algae can divide by multiple fission and spontaneously synchronize their cell cycle with the available light regime. The yields that can be obtained from a microalgal culture are directly affected by cell cycle events. Chromochloris zofingiensis is considered as one of the most promising microalgae for biotechnological applications due to its fast growth and the flexible trophic capabilities. It is intensively investigated in the context of bio-commodities production (carotenoids, storage lipids); however, the pattern of cell-cycle events under common cultivation strategies was not yet characterized for C. zofingiensis. In this study, we have employed fluorescence microscopy to characterize the basic cell-cycle dynamics under batch and continuous modes of phototrophic C. zofingiensis cultivation. Staining with SYBR green—applied in DMSO solution—enabled, for the first time, the clear and simple visualization of polynuclear stages in this microalga. Accordingly, we concluded that C. zofingiensis divides by a consecutive pattern of multiple fission, whereby it spontaneously synchronizes growth and cell division according to the available illumination regime. In high-light continuous culture or low-light batch culture, C. zofingiensis cell-cycle was completed within several light-dark (L/D) cycles (14 h/10 h); however, cell divisions were synchronized with the dark periods only in the high-light continuous culture. In both modes of cultivation, daughter cell release was mainly facilitated by division of 8 and 16-polynuclear cells. The results of this study are of both fundamental and applied science significance and are also important for the development of an efficient nuclear transformation system for C. zofingiensis.