Since the interspecific competition represents a critical issue for biodiesel production in open mass cultures of microalgae, laboratory studies aimed to select competitive species receive significant interest. In this work, a laboratory approach based on the method of multisizing cell counting was developed to monitor the competition course of single algal species grown in mixed cultures. Two marine microalgae, Dunaliella tertiolecta and Phaeodactylum tricornutum, were used to set-up an induced-contamination assay, with the aim to test their competitive capabilities. The evaluation of the oil production by dried biomass extraction was coupled to the growth assay to investigate the effect of competition on oil yield. The adopted experimental approach revealed effective as a method to selectively measure the algal growth of single species in mixed cultures, enabling to evaluate the competitive properties of Dunaliella outgrowing the contaminant species Phaeodactylum. At the end of the experiment the dominant species contributed more than 90% to the total biomass, while no loss of oil production was observed, the oil yield in the mixed being even higher than in the unialgal culture (3.15 vs. 2.28 mg). The outcomes of the induced competition suggest the use of Dunaliella as a competitive oil-producer species and especially support the potential of the experimental approach to be used for preliminary screening to drive species selection for open mass cultures.
Microalgae biomass is currently considered the most promising alternative as oil source for biodiesel production [
A key drawback of the outdoor systems is that, due to direct exposure to the atmosphere, the open mass cultures revealed susceptible to contamination by autochthonous undesired microalgae which can outcompete the inoculated strains [
The early studies on biodiesel production from microalgae, undertaken over the past few decades under the Aquatic Species Program [
The marine species Dunaliella tertiolecta and Phaeodactylum tricornutum have been maintained in our laboratory according to EPA standard procedures [
While both algae are oleaginous species, the oil content on biomass dry weight of Dunaliella is reported in a range of values (20% - 63%) higher than Phaeodactylum (20% - 30%) [
Based on the above described characteristics of the selected species, Phaeodactylum was used to contaminate Dunaliella. The interspecific competition was induced by the experimental addition of exponentially growing cells of Phaeodactylum (2200 cells/ml) to the unialgal culture of Dunaliella at the incubation day 2. Phaeodactylum cells were added by a ratio of 1:20, just to simulate a contamination event. The mixed culture was paralleled by unialgal control cultures of each species. All culture flasks were incubated under the same condition of 20˚C ± 1˚C, 14 h:10 h light:dark, cool-white fluorescent tubes (5000 lux), manual shake twice a day. The algal growth, as cell density (cells/ml), the biomass volume (µm3/ml) and the growth rates, as doublings per day (d/day), were measured until the stationary phase was reached in all the incubated cultures.
The algal growth measurement was performed by using the electronic particle counter (mod. Beckman Coulter MultisizerTM3). The technique is based on the Coulter Principle, also known as Electrical Sensing Zone method, allowing particles homogeneously suspended in a conducting solution to be simultaneously counted and sized. The counting system is thus able to accurately measure the cell number, as electrical pulses, as well as the volume of each drawn cell, within a wide size range. Aimed to define the specific size ranges of Phaeodactylum and Dunaliella a large number of preliminary analyses were performed on single cultures. After that, the analyti- cal range appropriate to cover the two different size distributions was defined and used to perform the cell count of all cultures. The selected range 10 - 300 µm3 was adopted since just enabling to discriminate the growth of the single species in mixed cultures.
The final biomass produced by contaminated and uncontaminated cultures was simultaneously harvested by centrifugation at the end of the experiment, hence dried in a thermostatic oven at 70˚C. The dry weight of biomass was gravimetrically measured and expressed as milligrams. The oil content was solvent-extracted from the dried biomass by homogenization in propan-2-ol:cycloexane:water 8:10:11 by volume [
The results of the preliminary analyses of unialgal cultures of Phaeodactylum and Dunaliella performed by multichannel counting to define their specific size ranges are shown in
Due to the Digital Pulse Processing technology of the instrument (Beckman Coulter MultisizerTM3), two distinct size-distributions, each one divided into 300 size channels, have been clearly defined for the two species, the MCV being 72 µm3 and 166 µm3, for Phaeodactylum and Dunaliella, respectively. As a result, we could select narrow ranges of interest respectively below and above 107 µm3 to discriminate the growth of the two single species when mixed in the competition experiment cultures.
The evolution trend of both species in the cultures was studied over 19 incubation days of competition assay. In
First of all, the fact that the Multisizer counting revealed able to simultaneously count the single species until the end of the experiment is to be appreciated, when considering that in previous investigations using haemocytometer counting method to study competition, most of the experiments failed before the outcome of competition, even between the same species, just because of difficulties in counting individual species, mainly due to cell clumping [
The growth curve of Dunaliella in the mixed was similar to that of the control culture, the stationary phase reaching maximum cell densities of 2 and 2.2 × 106 cells/ml respectively, after two weeks of incubation. By contrast, the growth of Phaeodactylum was markedly different when comparing the control with the mixed culture, where the contaminant species, which was inoculated at the incubation day 2, stopped growing in few days, reaching stationary values of 160 × 103 cells/ml at the experiment day 6, revealing unable to compete against Dunaliella, which successfully kept growing like its control, reaching the stationary phase at the incubation day 13. It should be noted that, even if all the cultures were grown under the same nutrient conditions, the cell density achieved by Phaeodactylum in the control culture (4.4 × 106 cells/ml) at the end of the experiment was two-fold higher than Dunaliella in control or mixed cultures. This large difference observed on cell number can be definitely explained by the specific difference of cell volume, Phaeodactylum MCV being two-fold lower than Dunaliella MCV. Actually, when considering the biomass volume, as measured by the particle counter, the much similar values of 350 × 106 vs. 320 × 106 µm3/ml were respectively found in Dunaliella and Phaeodactylum single cultures.
The invasive capacity of the contaminant species Phaeodactylum is revealed only when comparing the growth rates of individual species, as measured in the contaminated culture.
The drop in cell doubling shown after the experimental day 6 can likely be due to the limitation of nutrient resources. According to the distinctive properties of the species, as reported by previous studies [
The data obtained by means of the counting and sizing method were used to calculate the percent contribution of single species to the total biomass in the mixed culture, as plotted in
The increasing trend of Phaeodactylum reached the maximum contribution of 23% to the total cell number versus 12% of biomass at day 6. While the contribution of Dunaliella was decreasing from 100% to 77% of the
total cell number within 6 days, the chlorophyte progressively recovered its position of absolute dominance, with a final contribution up to 93% of total cells, and up to 97% of total biomass, at the end of the experiment, that is when the biomass was harvested, and the oil extracted, from all the cultures. In the case of the mixed culture the percent contribution of total yield was used to calculate the biomass and the oil yield produced by individual species. All the values of biomass and oil yield are shown in
As expected on the basis of the trophic potential of the culture medium, similar values of total biomass yield were measured in the cultures, in the mixed being even slightly higher than in the uncontaminated control, where Dunaliella was growing alone. In particular, the reliability of the multisizing counting method is highlighted when considering that similar values of biomass of the unialgal cultures of Dunaliella and Phaeodactylum, in spite of their great differences in cell number, were found either when gravimetrically measured as biomass yield, or by electronic counting as biomass volume. Furthermore, against the large difference found for Phaeodactylum when comparing individual species in the mixed with their unialgal controls, the much similar values of 8.83 and 8.59 mg found for Dunaliella confirm its undisturbed and absolute dominance in the competition and mainly support the capacity of the adopted counting technique to discriminate the contribution of single algal species to the total biomass in mixed cultures.
Likewise, very close values of oil yield were found in the unialgal cultures of Dunaliella and Phaeodactylum, while the oil production in the mixed culture was not lowered by the induced competition, being instead higher than in the uncontaminated control culture. The oil content, calculated as percent oil of the biomass yield, revealed a good agreement of all the specific oil production with literature data. Apart from the oil contribution of Phaeodactylum in the mixed culture (0.1 mg), which was regarded as negligible, the value of 26.16% of biomass, calculated in its unialgal control is just falling in the middle of the 20% - 30% range reported by the literature [
Culture | Species | Biomass (mg) | Oil yield (mg) | Oil content (%) | |||
---|---|---|---|---|---|---|---|
Unialgal | Dunaliella | 8.59 | 2.28 | 26.54 | |||
Phaeodactylum | 7.99 | 2.09 | 26.16 | ||||
Mixed | Dunaliella | 8.83 | 3.05 | ||||
Phaeodactylum | 0.29 | 0.10 | |||||
Total | 9.12 | 3.15 | 34.54 | ||||
[
In any case, instead of a loss of the overall productivity, the obtained results have rather pointed out the in- crease of oil production in the mixed culture, as an effect of the induced competition. It seems in agreement with recent literature supporting that the enhancement of the oil production can be linked to the microalgal species diversity of mixed cultures. The same authors are even suggesting that multispecies algal communities, instead of single species, should be cultivated in open-pond mass cultures to store more energy as lipids for biofuel pro- duction [
The adopted laboratory approach coupling the experimental design of induced competition with the instrumental multisizer-counting method proved effective showing high efficiency of Dunaliella, which exhibited competitive abilities to resist to the contamination of Phaeodactylum and to produce as high biomass in mixed as in unialgal culture condition. Also the oil production, which was not affected by the contamination event, supported the suitability of Dunaliella to be used in open mass-cultures for biodiesel production.
The use of the multichannel analyzer proved to be a sensitive and reliable method, capable to discriminate between two single species growing in mixed cultures, even at high cell-density levels, thus enabling to follow the evolution trend of competition as well as the calculation of the specific contribution to the total, biomass and oil yield, in mixed cultures. The obtained results strongly supported the adopted counting method as an effective strategy to study the interspecific competition between oil-producer algal species.
The adopted experimental approach supports the potential of laboratory algal assays for preliminary screening to drive species selection, in terms of their competitiveness, for their potential use in outdoor systems. The use of this approach is recommended as useful means to prevent the risk of lowering the oil yield which is possibly associated to the interspecific contamination in open mass-cultures for biodiesel production.