New technologies from the bioworld : selection of biopolymer-producing microalgae

Microalgae are studied because of their biotechnological potential. The growth of microalgae aims at obtaining natural compounds. Due to the large amount of accumulated polymer waste, one of the solutions is the use of biodegradable polymers. The objective of this work was to select biopolymer-producing microalgae and to study the cell growth phase in which maximum production occurs. Microalgae Cyanobium sp., Nostoc ellipsosporum, Spirulina sp. LEB 18 and Synechococcus nidulans were studied. The growth was carried out in closed 2 L photobioreactors kept in a chamber thermostated at 30 °C with an illuminance of 41.6 μmolphotons.m -2.s-1 and a 12 h light/dark photoperiod. The biopolymers were extracted at times of 5, 10, 15, 20 and 25 d. The microalgae that had the highest yields were Nostoc ellipsosporum and Spirulina sp. LEB 18 with crude biopolymer efficiency of 19.27 and 20.62% in 10 and 15 d, respectively, at the maximum cell growth phase.


Introduction
Cyanobacteria were the first phototrophic organisms capable of producing oxygen.They are responsible for the conversion of Earth's atmosphere from anoxic to oxic [1] .For the production of biomass with specific characteristics, manipulation of the culture conditions is a key factor [2] .
Bacteria and cyanobacteria have the capacity to produce polyhydroxyalkanoates (PHAs) [13,18] , which are biodegradable polyesters with potential use as polymeric materials [19] .Biodegradable polymers are alternative replacements for petrochemical polymers [20] .
Reducing the consumption of plastic materials is difficult because of their versatile properties.However, it is possible to replace the petrochemical polymers with alternative materials that have similar polymer properties but show rapid degradation after disposal [20] .
PHAs may positively change the scenario of global climate impact by reducing the amount of non-biodegradable polymers used [20] .Mixed cyanobacterial and bacterial cultures to produce PHAs are emerging due to the potential residuary use for growth and low installation cost towards a profitable production of polyhydroxyalkanoates.The growth of microalgae does not require large amounts of land and can occupy areas unsuitable for agriculture, thus not competing with food production, due to the possibility of using photobioreactors that maximize biomass production [21,22] .
The objective of this work was to select biopolymer-producing microalgae and to study the phase of cell growth in which maximum production occurs.

Culture conditions
The cultivations were performed in closed 2 L photobioreactors with a working volume of 1.5 L and continuous agitation by the injection of sterile air to avoid the precipitation of the biomass.For Nostoc ellipsosporum, Spirulina sp.LEB 18 and Synechococcus nidulans, the initial concentration was 0.15 g.L -1 , but for Cyanobium sp., the initial concentration was 0.2 g.L -1 .The triplicate cultures were kept in a thermostated chamber at 30 °C for 5, 10, 15, 20 and 25 d, for a total of 15 experiments for each microalgae.The illuminance used was 41.6 μmol photons .m - .s - with a 12 h light/dark photoperiod maintained by 40 W fluorescent lamps.

Analytical determinations
Daily samples were collected aseptically for the monitoring of the cell concentration and pH.Cell concentration was determined by optical density at 670 nm in a spectrophotometer (Quimis Q798DRM, Brazil) with a calibration curve relating the optical density to the dry weight of the microalgal biomass [27] .The pH determination was performed in digital pH meter (Quimis Q400H, Brazil) following AOAC methodology [28] .

Determination of the crude biopolymer yield
The crude biopolymer yield (Y CB ) was calculated according to Equation 1, where C cbt is the concentration of crude biopolymers (g.L -1 ) at time t (d), C cb5 is the concentration of crude biopolymers (g.L -1 ) at time 5 d, t is the time (d), and t 5 is the time at 5 d.

Extraction of crude biopolymers
After 5, 10, 15, 20 and 25 d of experiment, the cultures were centrifuged at 7500 rpm for 20 min at room temperature (Hitachi, Japan) to separate the wet biomass from the biopolymer of the culture medium.Later, for every 1 g of dry biomass, 100 mL of distilled water and 25 mL of sodium hypochlorite (10-12% active chlorine (w/v)) were added to the wet biomass, and the solution was kept under stirring for 10 min.The resulting suspension was centrifuged (7500 rpm for 20 min at room temperature).Then, the supernatant was discarded, and the precipitate was washed with 100 mL of distilled water.The sample was centrifuged again, and the supernatant was discarded.This process was repeated adding 50 mL of acetone.The final precipitate (crude biopolymers) was dried at 35 °C for 48 h.The efficiency (η) of crude biopolymers in relation to microalgal biomass (%) was calculated using Equation 2, where m cb is the final mass of crude biopolymer obtained from the microalgal biomass (g), and m ma is microalgal biomass (g).

Statistical analysis
The results were processed by analysis of variance (ANOVA) and Tukey's test to compare the means of the parameters analyzed with a 95% confidence level.

Results and Discussions
The growth curves of cyanobacteria Cyanobium sp., Nostoc ellipsosporum, Spirulina sp.LEB 18 and Synechococcus nidulans (Figure 1) showed different behaviors in spite of each species having its own specific growth characteristics and different culture media.In preliminary tests, it was observed that when the microalga Cyanobium sp. was grown at low biomass concentrations (0.15 g.L -1 ), it showed photoinhibition in its growth; therefore, the assays were carried out with an initial biomass concentration of 0.2 g.L -1 , thereby preventing cell death and providing the lag phase of growth.
Spirulina sp.LEB 18 (Figure 1c) showed early stationary growth phase after 20 d of culture.For Cyanobium sp., Nostoc ellipsosporum and Synechococcus nidulans, the stationary phase of growth was not observed by the end of the 25 d of culture.To verify the growth phases of the microalgae Cyanobium sp., N. ellipsosporum and S. nidulans, it would be necessary to grow the cultures for a longer period.For large-scale production, such a long culture period is impractical for the production of biopolymers.Sharma and Mallick [29] cultivated Nostoc muscorum microalgae in BG-11 medium with a phosphorus deficiency and addition of exogenous carbon sources and found an increase in the production of PHB.Yields of up to 8.6% (PHB) were found when the extraction of the polymer was performed in the early stationary phase of growth of the microalgae (21 d of culture), whereas in log phase, the yield was 6.1%.Samantaray and Mallick [12] cultivated the microalga Aulosira fertilissima during 14 d and observed an accumulation of 6.4% of PHB at the end of logarithmic growth phase.
The microalga Nostoc ellipsosporum presented a different behavior in its cell growth compared to the other microalgae under study.During the first 8 d of culture, it showed cell growth, then ceased and remained constant until the 17th d, after which it presented new cell growth.This growth pattern may have occurred because when the microalgae are under a particular nutrient limitation, they use a substrate from its own cell as a nutrient, enabling continued growth.If there is a lack of carbon, the microorganism can consume the biopolymer itself.In this case, it is believed that the biopolymer may have been consumed, because after the 10th d of cultivation, the yield of biopolymers was reduced (Table 1).Another nutrient that may have had an influence was nitrogen, whose release in the culture medium from amino acids of phycobiliproteins and chlorophyll can possibly allow cell maintenance to occur [30,31] .
The cyanobacterium Nostoc ellipsosporum presented a cell concentration less than the others but had higher efficiency (Table 1) and crude biopolymer yield (Table 2).
Among the microalgae under study, Nostoc ellipsosporum and Spirulina sp.LEB 18 stood out.These microalgae showed the higher efficiency of crude biopolymers (PHB) and did not differ significantly (p<0.05) each other from 15 d.However, Nostoc ellipsosporum reached a crude biopolymer   2).Panda et al. [15] found that the cyanobacterium Synechocystis sp.PCC 6803 accumulated biopolymer PHB in its cells.It has been found that when cultured in BG-11 medium under phosphorus and/or nitrogen deficiency with the addition of exogenous carbon sources, this microalgae showed a higher yield (4.5%) of PHB in the early stationary growth phase (at 21 d cultivation), while in the logarithmic phase, the yield was 2.9%.
The microalga Cyanobium sp. did not achieve significant results (p>0.05) for the production of crude biopolymers.The cyanobacterium Synechococcus nidulans showed the highest PHB efficiency (11.01±1.49%)at a greater time of growth (25 d) in relation to the microalgae Spirulinasp.LEB 18 and Nostoc ellipsosporum.Therefore, its use is less interesting compared to Nostoc ellipsosporum and Spirulina sp.LEB 18. Lower yields (3%) of PHB were found by Sankhla et al. [32] in the stationary phase of growth when studying the production of PHB by Brevibacillus invocatus MTCC 9039.
The lowest yields obtained in culture times greater than 10 d (Nostoc ellipsosporum) and 15 d (Spirulina sp.LEB 18) may be due to the depletion of nutrients from the medium, especially carbon, which leads to consumption of the biopolymers for cell growth and maintenance.The results showed the effect of culture time on the production of biopolymers.This difference in yield is associated with the fact that the production of the polymer depends on the availability of the source of carbon and energy, which vary as a function of the culture time.Bhati and Mallick [13] studied the microalga Nostoc muscorum for the production of PHB-HV with yields of 16.6% in 10 d of incubation.For the same microalga, yields of different biopolymers were observed at different times using different carbon sources.When BG-11 medium was used with the addition of propionate, the highest yield was 12.6% in 21 d and 16.6% in 10 d with the addition of valerate.The highest yields were in the late exponential phase of growth.Mallick [33] studied the production of PHB-HV in Nostoc muscorum using BG-11 medium with the addition of propionate yielding 28.2% of biopolymer in 14 d of culture (late exponential growth phase).
Several microalgae, especially cyanobacteria, are able to accumulate intracellular biopolymers, especially poly-3-hydroxybutyrate and poly (3-hydroxybutyrateco-3-hydroxyvalerate) belonging to the group of polyhydroxyalkanoates.By modifying the culture conditions, particularly the nutrients, one can divert the metabolic pathways, causing the microorganism to synthesize larger amounts of biopolymers.
Studies are being carried out with photosynthetic mixtures of bacteria and algae that accumulate PHA in conditions with different concentrations of nutrients, and these studies have achieved PHB yields of 20%.The use of mixed photosynthetic culture (bacteria and microalgae) has emerged as an alternative system for the production of PHA, potentially minimizing feed costs through the use of solar energy [34] .
The defatted biomass of microalgae Dunaliella tertiolecta was used for the production of biopolymers in different salt concentrations, obtaining a yield of 82% [11] .High yields of biopolymers can be achieved using microalgae.It is possible to conclude that many microalgae are able to intracellularly accumulate PHB granules.However, different behaviors are observed due to the use of different microalgal sources and concentrations of nutrients and growth conditions.

Conclusions
This study showed that in order to produce biopolymers from microalgal cultures, the microalgae Spirulina sp.LEB 18 and Nostoc ellipsosporum would be the best candidates.Both microalgae had higher concentrations of biopolymers at short growth times (Spirulina sp.LEB 18, 20.62% in 15 d; Nostoc ellipsosporum, 19.27% in 10 d).Combining the growth of microalgae and biopolymer production is a strategy with the potential to significantly reduce environmental pollution problems, through both the use of industrial waste as a source of nutrients for the culture medium and the replacement of petrochemical origin polymers by biopolymers degradable and compostable when disposed of in the environment.

Table 1 .
CrudeFor the same letters, the averages do not differ significantly (p<0.05) by Tukey test.Lowercase letters compare the results in columns.Uppercase letters compare the results in the rows.*Values correspond to averages of results obtained in triplicate with their respective standard deviations.efficiency of 19.27% in 10 d and Spirulina sp.LEB 18 reached 20.62% in 15 d of culture.The crude biopolymer efficiency of Nostoc ellipsosporum was 2.05 g.L -1 .d - at 10 d, where as that of Spirulina sp.LEB 18 was 1.48 g.L -1 .d - at 15 d (Table biopolymer efficiency (%, w/w*) for microalgae at different culture times.