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Lipids productivity of cyanobacterium Anabaena vaginicola in an internally illuminated photobioreactor using LED bar lights – Scientific Reports

Microbial culture and maintenance

A. vaginicola isolated from rice (Oryza, sativa L.) was obtained from the previous study and all the necessary methods used in this study were in accordance with the relevant guidelines13. BG11 medium which contains synthetic nitrogen and carbon sources and other inorganic salts (NaNO3 and Na2CO3), was used to support growth of the test cyanobacterium14. Light is the driving force for photosynthesis and details of all these growth requirements and the cultivation process are given elsewhere11,15. Briefly, BG11 medium poured in several graduated cylinders, was inoculated with use of A. vaginicola. By installation of daylight fluorescent light lamps in horizontal position in front of graduated cylinder, the culture was illuminated, and a lux meter was used for measuring light intensity (LM 76 Light meter, Multimetrix®, China). The light at level of 80 µmol photons m−2 s−1 was provided. With use of laboratory tubing, CO2 was supplied through continuous aeration with filtered wet air (Fig. 2).

Figure 2
figure 2

Inoculation of BG11 medium in graduated cylinder with A. vaginicola– the grown bacterium was used for the IIPBR study.

Design, geometry, and operation of IIPBR

Structural details of the experimental setup (600 ml as the nominal volume, 13 cm as height and 8 cm as the diameter) are presented in Fig. 3.

Figure 3
figure 3

Schematic diagram showing experimental setup of the IIPBR schematically shown (A) see below for the details. The photograph of the photobioreactor which has been placed inside the incubator, is also presented (B). The dimension details of LED bar light which was placed in the Pyrex glass tube presented schematically (C). The photograph of temperature control switch sensor module is also given (D). (A) 1-main body of the IIPBR, 2-internal illumination system with use of the LED bar light positioned inside the Pyrex glass, 3-aquarium pump providing fresh air, 4-warm air outlet, 5-electrical power connection, 6-water container, 7-aquarium pump and relevant air filter, 8-sampling port, 9-magnetic stirrer and magnetic stir bar, 10-incubator, 11-system’s temperature controller, 12-top view of the IIPBR—See the text for the details.

illumination details were as follows: Two 8 W LED bars (dimensions 30 × 1 cm) were used for the illumination where one bar was able to emit cool white light (6500 K) and the other one was capable to emit warm white light (3500 K), and both were connected to a 220 V AC power supply. Each of the LED bar consisted of 72 diodes with 180° beam angle (Tranyton Co. Ltd., Taiwan). LEDs were placed inside a Pyrex glass tube of 22 mm internal diameter. The temperature of an LED illumination system is higher than the ambient temperature (i.e., part of electric energy is converted to heat energy) and is advisable to use an appropriate system for controlling temperature. This adjustment simply was done by use of an aquarium pump (ACO-5505, HAILEA®, China) where fresh air would be available through connecting tubing (Fig. 3). For keeping temperature of the Pyrex glass surface at 28 °C, air flow rate was adjusted. Incubator made of chipboard (10 mm thickness), has the following dimensions (L* W* H): 50* 50* 50 cm. Electric coiling and fans were places at two positions and by fixing a temperature control switch sensor module at the top corner of the incubator (XH-W1401, Covvy®, China) each of these was able to respond appropriately to the temperature fluctuations. With all these considerations, the setup was finalized by placing the IIPBR inside the incubator and in this manner fluctuations of the system’s temperature were kept at the minimum level (± 1°C).

CO2 as the substrate was admitted into the IIPBR using an aquarium pump. Carbon dioxide was quantified in terms of aeration rate where air was passed through filter unit (0.22 μm) and water container (250 ml flask) (Fig. 3). Air flow rate was monitored by a flow meter (LZB-3WB, Changzhou Chengfeng® Flowmeter, China). Wet air provides a favorable environment for growth and metabolic activity.

Table 1 presents the details of the experimental plans and the growth behavior was tested in terms of A. vaginicola expression to certain environmental stresses, i.e., Nitrogen content of the BG11 medium (0.5, 1.5 g/l as NaNO3 and the system without inorganic Nitrogen), quality and quantity of LED light bar (warm and cool white LED with intensity equaled to 80, 150, and 8 µmol photons m−2 s−1), and amount of CO2 expressed in terms of air flow rate (120, 40 ml/min, and system without aeration).

Table 1 Experimental plan used in the IIPBR considering different regimes of intensities of the LED light bar, inorganic nitrogen content of the growth medium, and amount of CO2 admitted to the reactor.

A. vaginicola culture grown in graduated cylinder as described in Sect. 2.1, was used for the IIPBR study where the inoculum size was 4 ml/l considering 400 ml as the IIPBR working volume. IIPBR operation lasted for 11 days, and analyses were performed regularly (d−1) by taking appropriate sample in each time interval.

Analytical methods

The data were presented properly, using one-way analysis of variance (ANOVA test- Microsoft Excel 2021). The level of significance was set at α = 0.05 (i.e., type I error- accepting the alternate hypothesis when the null hypothesis is true) and 95% of the reported value lies between ± 3 SD (standard deviation).

Growth of the cyanobacterium was determined spectrophotometrically in the rage of 380 to 800 nm (V-550 UV/VIS Spectrophotometer, JASCO®, Italy) where the absorption peaks were used for quantification. Gravimetric method was used to measure biomass dry weight, i.e., the cells were collected daily using 10 ml of sample and by Büchner funnel liquid portion was separated and the wet residue remained on the funnel surface (Whatman filter paper 4) was dried in a laboratory oven (105°C for 24 h). The data fitting was performed using regression analysis.

A further note was to extract lipids from the test cyanobacteria and the obtained content was estimated15,16. Different amounts of chloroform and methanol were used at the dried biomass and homogenization and filtration were carried on according to the details given in the relevant reference. The Chloroform solvent was let to evaporate, and the residue was used in the gravimetric methods and the lipids content was determined.

Growth kinetics and its relationship to lipids production

A typical growth curve for A. vaginicola was obtained using exponential growth model:

$${C}_{t}={C}_{i}{e}^{mu .t}$$

(1)

where Ci is the biomass content (g L−1) at initial stage, Ct is biomass at any time t during experiment, and µ is the growth rate constant (d−1). For measuring the µ, the linearized form the Eq. 1 was used:

$$mu =left(ln {C}_{t}-mathit{ln}{C}_{0}right)/t$$

(2)

With use of the growth rate constant, biomass productivity was determined (mg L−1 d−1):

$${P}_{B}={C}_{t}times mu $$

(3)

Capacity of A. vaginicola for production of lipids was determined in terms of lipids yield (%):

$${Y}_{L}=left({W}_{L}/{W}_{d}right)*100$$

(4)

where WL is the weight of the total lipids (g), and Wd is the weight of the dry biomass (g).

Further approach on A. vaginicola was to measure lipids productivity (mg L−1 d−1) based on the following equation:

$${P}_{L}=left({P}_{B}*{Y}_{L}right) / 100$$

(5)

IIPBR energetics

Considering different forms of energies which participate in functionality and operation of these types of photobioreactors, is a reasonable approach for giving an estimate for the process cost as described in the results and discussion section. The point of interest in the present study is IIPBR’s energy utilization in terms of energy of the LED light bar and energy consumed for mixing operation as the input energy. Biomass productivity and the lipids formation by the A. vaginicola cyanobacterium culture under selected operation variables described in the experimental (Table 1) were measured.

On the bases of energy required for system’s illumination (EL- W m−3), IIPBR performance was assessed, and comparison was made with other types of the photobioreactors (PBRs). EL was estimated in terms of power input per unit culture volume17:

$${E}_{L}=frac{0.22{I}_{o}A}{V}$$

(6)

where Io is incident light intensity per unit incident area (µmol m−2 s−1), A is incident area (m2), and V is the culture volume (m3).

Similar approach was used for estimation of mixing energy input per unit culture volume (EM,B-W m−3)18:

$${E}_{M,B}=frac{Qgamma h}{60V}$$

(7)

where, Q is the volumetric gas flow rate (m3 min−1), γ is the specific weight of the broth (N m−3), h is the culture depth (m), and V is culture volume (m3).

On bases of total energy used for IIPBR performance, biomass productivity per unit power input (PUV-g W−1 d−1) was estimated and used for further comparisons with other photobioreactors19:

$${P}_{UV}={P}_{B}/({E}_{L}+{E}_{M,B})$$

(8)