FOCUSING ON DRAWBACKS IN MANAGING THE
COMPLEXITY OF MICRO-ALGAL COMMUNITY
Rossana SIDARI and Andrea CARIDI
Department of AGRARIA , Mediterranea University of Reggio Calabria, Loc. Feo di Vito,
89124 Reggio Calabria, Italy
Different autochthonous microalgal mixed blooms
Mixed bloom observed
with magnification of 300x
Mixed bloom observed
after the zymolyase treatment
Commercial kit for
cryostorage
Mixed bloom
Introduction
Nowadays the attention of researchers is paid on the extreme specialization of microbial pure cultures. The simplification of the microbiological actors playing in a process has been considered the way to gain the best results in producing foods, beverages, and industrial products. Microalgae are involved, among others, in the production of alternative sources of energy. The majority of literature concerns study and selection of pure microalgal strains (Abou-Shanab et al., 2011; Amaro et al., 2011; Sydney et al., 2011; Duong et al., 2012; Lim et al., 2012) using classical methodologies of observation, counting, isolation, and storage. A few studies shift the attention from pure cultures to microalgal communities obtained mixing different microalgal pure cultures, also in the presence of other microorganisms (Phatarpekar et al., 2000; Papone et al., 2012).
Drawbacks in studying mixed microalgal blooms
Studying naturally evolved seawater mixed microalgal blooms, the structure of the stable population established was a hurdle in studying this complex microalgal community. In addition, being mixed microalgal bloom a natural community evolved during times, it is composed not only by different species of microalgae but also by marine bacteria that contribute to the aggregation phenomenon. Anyway, the microbial complexity has a key role in maintaining a stable state with capacity to adapt to environmental changes (May, 1977); moreover, the interaction among the different species composing a stable population can lead to an improvement of the culture performances. Different approaches are described in order to characterize and collect mixed microalgal blooms.
Bloom quantification by spectrophotometer; bloom characterization by microscopy
All the existing microalgae identified by microscopy are well known and reported for their high lipid accumulation capabilities. The ratios of saturated fatty acids (SFA) and unsaturated fatty acids (USFA) were also evaluated (Venkata Mohan et al., 2011).
Algal growth, i.e. biomass growth, was measured with a spectrophotometer at 600 nm, using deionised water as the blank control. Approximately 3.5 ml of each sample was used and measured in 1 cm cuvettes. Flasks were agitated by hand prior to measurement in order to prevent settling of the algae. After 14-21 days of cultivation the predominant groups of algae species in each flask were characterized by conventional microscopy (Odlare et al., 2011; Krustok et al., 2013).
It is important to consider that the use of the spectrophotometer to monitor mixed blooms presenting aggregates is negatively affected by reading instability due to the aggregates sedimentation. Bloom preservation at -80°C after centrifugation
Algal biomass was harvested from the media by centrifugation and thoroughly mixed to account for potential variability in algal lipid content between the three reactors. From the mixed biomass, five samples were removed and lyophilized to determine the average moisture content of the harvested algal biomass, based on the mass of water removed. The lyophilized algal biomass was then properly stored for later testing. The remaining centrifuged algal biomass was immediately preserved as centrifuged at -80°C (Sathish and Sims, 2012).
It is important to consider that the bloom preservation is restricted only to carry out further analysis but not to collect microalgal species due to the absence of cryoprotectants. Bloom evaluation by indirect methods
Algal bloom evolution was evaluated by pigment measurements (phycocyanin, chlorophyll a and b), determination of photochemical and metabolic activities, and determination of the concentrations of total nitrogen and total phosphorous (Jia et al., 2013).
It is important to consider that this approach does not give adequate information about mixed microalgal blooms.
Aim
This contribution aims to draw attention to the complexity in studying mixed marine microalgal populations naturally evolved in order to stimulate new research and improve microalgae management.
Materials and methods
Seawaters - collected along the coast of Reggio Calabria (South Italy) - was filtered by a 100-μm net, enriched with Walne medium (1 ml/l) plus vitamins (0.1 ml/l) (Walne, 1996) and maintained at natural temperature and photoperiod. The samples were daily observed by an optical microscope (Zeiss standard 20) from sampling date until flourishing of the microalgal population. Throughout the evolution, changing consisting in appearing of some species and disappearing of others until the establishment of a stable mixed populations were observed. The different species clumped together forming macroscopic and persistent aggregates that impaired managing and studying of the mixed microalgal blooms. In fact, the cell aggregates do not allow the easy identification of the different type of cells by classical keys, the easy achievement of pure cultures by micromanipulation, the determination of growth curves for each population present in the culture - due to the drawbacks encountered in using cell counting chambers, and the cryostorage - using commercial systems - due to the structure of the sample. Starting from the consideration that the cell aggregates could be due also to exopolysaccharides produced by the cells themselves, the idea was to attack these substances by enzymatic treatment. Zymolyase, was used at the concentration commonly used to lysate the yeast ascus (20 U/ml). So, the culture was added with the enzyme and observed before the treatment and after 30 min. A trial of cryostorage of the mixed bloom tested using a commercial kit was carried out. This system - beads in a solution of cryoprotectans - foresees a first step of contact between culture and beads plus cryoprotectant solution to allow the cells to adsorb to the beads and a second step of discarding the solution before cryostoring the culture at -80°C (data submitted for publication).
Results and perspectives
The use of zymolyase allowed to partially disaggregate the cell macro-aggregates with positive effect on the microscopic structure of the bloom. This allow to improve the observation of the species composing the culture; according to morphological features it was composed by Pseudoanabaena sp., Nodularia sp., Anabaena sp., Cymbella sp., Nitzschia sp., and Navicula sp. This approach partially solve drawbacks in studying mixed microalgal blooms but still remain the impossibility to use growth chambers. Due to the presence of cell macro-aggregates, the commercial kit for cryostorage was not useful; in fact, the persistent aggregates remained in suspension in the solution of cryoprotectans and were discarded together with the solution not guarantying the proper cell attachment to the beads.
Some proposals, in order to further improve the managing of this type of cultures may be: a) use of mechanical treatments; b) use of different enzymes alone or in mixture; c) use of different cryoprotectans alone or in mixture.
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