Glass jars filled with pond water algae with their rim open as control and test with rim sealed with AIRplus® pillow plastics showed some peculiar results. On day 1, the volume of pond water in both the glass bottles was same. It was observed that even up to the 50th day, algae remained green in both the closed glass bottle as well as open glass bottle. However, it was seen that there was no water loss in the bottle sealed with bubble wrap plastic while about 50% water loss occurred in an open glass bottle (Figure S1). However, there was full water retention in the sealed jar, with roughly equal algal growth in both. This proved that the sealing of glass bottles' rims with bubble wrap plastic didn’t starve the algae for exchange of gases (CO2 and O2) needed for photosyntehsis and respiration, nor let the water evaporate. Since one of the major drawbacks of open raceways is water loss by evaporationa and contamination (Gutiérrez et al., 2016; Moheimani & Borowitzka, 2006), the sealing of even an open raceway by large sheets of bubble wrap plastic could probably reduce these problems, even without putting the algae inside the bubbles.
Our quest was to quantitatively check the CO2 and O2 availability and water retention of different bubble wrap plastic materials (NP, LDPE, HDPE) and control by recording physical parameters such as humidity, pH, conductivity (µS), and TDS.
It was observed that the pH with all bubble wraps as well as control was alkaline, reaching ~ 12 at the 20th day and decreased a little in control on 20th day (11.3 ± 0.25 µS) (Fig. 1A). It was. however, seen that there were only minor changes in the conductivity and TDS value in all bubble wrapped test samples. Conductivity decreased in controls from 5.64 ± 0.02 µS to 2.42 ± 0.32 µS from the 1st to 20th day; whereas in NP, LDPE and HDPE it increased from ~ 5.64 ± 0.10 µS on the 1st day reaching nearly 5.74 ± 0.25 µS in NP, 6.95 ± 0.24 µS in LDPE and 7.23 ± 0.27 µS in HDPE (Fig. 1B). However, the control sample without any bubble wrap showed constant decrease in its TDS value and reached from 5.64 to 1.61 ppt from 1st day till 20th day respectively (Fig. 1C). This was possibly because of high availability of CO2 and other gases. This was unlike the NP, LDPE and HDPE samples where the water was turbid as there was constant increase in TDS as seen in Fig. 1C. However, the TDS was near 0.07 ± 0.04 ppt on the 1st day and increased to near ~ 2.5 ± 0.25 ppt on 20th day. Although it was least for LDPE bubble wraps at 2.12 ± 0.24 ppt.
The conductivity and TDS changes with the change in carbon dioxide absorption and desorption were concordant in pattern as explained by Hindi and Azizi(2020) in a bubble column reactor tank (Al-Hindi & Azizi, 2020). However, there was minor or almost no changes in pH which is concordant with the earlier reported studies for change of pH with CO2 absorbtion studies (Al-Hindi & Azizi, 2020). Increase in CO2 absorption results in increase in salinities with the effect of different water types studied for CO2 absorption and desorption studies in bubble column reactors (Al-Hindi & Azizi, 2020). Lastly relative humidity of the culture room where the experiment was conducted was stable for all days reaching between 15–17% which is a significant factor for the maintaining concentration of gases present in the atmosphere throughout the experiment.
The UV-Vis spectroscopic observations for different bubble wrap/s (NP, LDPE and HDPE) test tubes and control for CO2 absorption and desorption showed that the control test tube changed its turbidity after addition of calcium hydroxide solution into it. Figure 2 illustrates that on 5th day, control test tubes showed absorbance of 1.77 at 208 nm whereas NP, LDPE, and HDPE showed absorbance of 1.75, 1.73 and 1.72 at 212 nm respectively. As the time of exposure increased, absorbance of CO2 increased from 10th to 20th day. The highest absorbance (1.91) was reported in control sample on 20th day whereas there were significant changes in the absorbance in NP, LDPE and HDPE observed as 1.72, 1.68 and 1.67, respectively. The high absorbance of CO2 in control was due to the fact that maximum CO2 was absorbed forming CaCO3 precipitate (Gettens et al., 1974). This is clearly elaborated in Fig. 2D due to maximum area under curve occupied by control compared to rest of the bubble wrap/s plastics sealed test samples. It was further seen that the increase in CO2 absorption results in not only decrease in TDS but also decrease in absorption coefficient as is seen from the UV Vis spectral studies for control sample which is concordant with earlier reported studies(Al-Hindi & Azizi, 2020). Even though LDPE bubble wrap showed comparatively better absorption of CO2, all the bubble wrap/s were further tested for growth of diatom, water loss, chlorophyll content, cell density via absorbance, lipids and biofuel from diatoms
Henceforth, fixed number of diatom cells (~ 479 x 102 cells mL− 1) were inoculated in 100 mL modified f/2 medium(Vinayak et al., 2014) contained in 250 mL conical flasks sealed with NP, LDPE and HDPE bubble wraps alongwith control in triplicates. The experiment was run for 40 days which showed varied diatom cell count as shown in Fig. 3A. It was observed that control showed fall in diatom growth on the 40th day (197 x 102 cells mL− 1). The diatom growth decreased on 40th day in flasks sealed with NP (429 x 102cells mL− 1) and HDPE (856 x 102cells mL− 1), however, only LDPE, showed a stable quasi-exponential behaviour in growth of diatom cells until the 30th to 40th day (1152 x 102cells mL− 1). The growth condition was further verified by recording absorbance at 680 nm using UV-Vis spectrophotometer (Fig. 3B). On 10th day, LDPE sample displayed a maximum absorbance of 0.4 and maximum cell count (499 x102 cells mL− 1) in LDPE due to absorption of CO2 gases required for photosynthesis. However, at 40th day, lowest absorbance (0.052) was found in the NP samples having lowest cell count of 429 x102 cells mL− 1. In LDPE at 40th day, though there was maximum absorbance (0.084) with maximum cell count of 1152 x102 cells mL− 1. However, it was low in HDPE (0.018) with cell count of 856 x102 cells mL− 1on 40th day as seen in Fig. 3B. The results were in concordance with the theory that cell growth is directly proportional to the absorbance of light getting absorbed due to scattered particles in the cuvette (Ritchie & Sma-Air, 2020). Also the cell growth in LDPE was linearly proportional to the CO2 gases absorbed in LDPE samples as seen in Fig. 2.
On the offset the chlorophyll concentration at 10th day was highest for control sample (0.835 µg mL− 1) as compared to NP (0.456 µg mL− 1), LDPE (0.752 µg mL− 1) and HDPE (0.555 µg mL− 1) test samples inoculated with fixed number of diatom cells (Fig. 3C). The chlorophyll concentration of control increased till 20th day and decreased thereafter. On 20th day, chlorophyll concentration of control sample decreased to 0.735 µg mL− 1. Decrease in chlorophyll concentration in control sample after 20th day is because of water loss and microbial contaminations concordant with open raceway culturing results (Borowitzka & Moheimani, 2013). The chlorophyll concentration in LDPE and HDPE at day 40 reached to 3.34 µg mL− 1 and 2.5 µg mL− 1 respectively. The amount of chlorophyll content is also directly proportional to cell growth and the results obtained were in accordance with the cell count in each set. This was also in concordance with earlier research demonstrating a direct correlation between the chlorophyll content and cell count/growth(Groß et al., 2021; Myers & Kratz, 1955). The chlorophyll content constitutes the major carotenoids which are a group of high value metabolites being synthesized in a plant. The water content was measured for each sample on the 0, 10th, 20th, 30th and 40th day and it was found that there was no remarkable water loss in bubble wrap/s closed samples except control which displayed significant water loss (~ 37%) (Fig. 3D) and obvious microbial contamination. This is the reason closed system photobioreactors are superior to open raceway ponds but due to their high operational cost they have limited use(Gordon et al., 2019a).
Further it was found that the amount of lipid at day 10 was 26.17 µgmL− 1, 22.40 µgmL− 1, 25.09 µgmL− 1 and 25.97 µgmL− 1for control, NP, LDPE and HDPE respectively (Fig. 4A). Lipid content further increased substantially in LDPE 31.21 µgmL− 1 on 20th day. The control sample showed decrease in lipid contents (2.33 µgmL− 1) on 40th day which is possibly due to decrease in cell count and contaminations in open culture flask which was not wrapped with bubble wrap at its mouth rim. The fall in lipid was also observed in NP (10.24 µgmL− 1) and HDPE (15.83 µgmL− 1) on 40th day. However, lipid yield continued to rise in LDPE and was 33.88 µgmL− 1 and 34.89 µgmL− 1 on 30th and 40th day. Comparing these results showed that the maximum amount of lipid was found in LDPE on 10th day but thereafter HDPE showed a decline on 40th day in coherence with low cell count in HDPE. This was probably also due to the fact of scarcity in CO2 and thus decline in photosynthesis. Hence even though CO2 plays an important role in photosynthesis to synthesize carbohydrates required for its fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) enzyme in C4 pathway of cell photosystem(Riebesell, 2000). Its deficiency may ultimately result in failure to synthesize carbohydrates required for further energy generating pathways. Further when these lipids were transesterified they showed the formation of Diafuel™ (Biofuel from diatoms) i.e. fatty acid methyl esters (FAME)(Fig. 4B). Maximum Diafuel™ percentage was 37.27% per DW for the LDPE sample, less in 28.1 % per DW in HDPE and minimum for NP 2.35% per DW and least in control sample (1.6%) per DW on 40th day.
On the other hand, the diatoms stained with Nile red dye for neutral lipids on 0 and 40th day in LDPE showed neutral lipids stained diatoms by confocal microscopy (Figure S2). The optical images clearly showed that lipid in diatoms increased on 30th day when cultured in the LDPE sealed conical flask without requiring any extra post nutrient feed. Nile red staining has been used in analyzing neutral lipids in Phaeodactylum tricornutum and is one of the most reliable method for insitu testing for amount of neutral lipids in cells(Wu et al., 2014). The three dimensional plot of amount of neutral lipids stained in diatoms on 0 and 40th day as seen in Fig. 4C and D clearly shows increase in amount of neutral lipids in diatoms cultured in LDPE flask PBR. It indicates it to be the most efficient plastic bubble wrap material to seal PBR while simultaneously maintaining a closed microcosm avoiding contamination and water loss.
Since one of the major drawbacks of open raceways is water loss by evaporation and airborne and exotic species contaminations(Ashokkumar & Rengasamy, 2012). Therfore, sealing of open raceways by bubble wrap/s plastic sheets could alleviate these problems economically and in a facile manner.
Not limiting to our study on diatoms we wanted to study its on ground application on a LDPE bubble wrap material measuring 6” ×6” with each bubble having diameter of 0.25” was chosen measuring was chosen as a whole. Hence one of the green microalgae Haematococcus pluvialis rich in astaxanthin and on high demand in pharmaceutical and nutraceuticals industry was tested and inoculated in the LDPE bubble wrap for 21 days. However, using bubble wrap having a bubble diameter of 0.25” made cell counting cumbersome due to frequent puncturing of the bubble wrap. Hence cell density was measured by histogram of each photographed image spilt into red, green and blue processed via Image J software. Figure 5 shows the blue channel of the image shifted towards a hike in its mean value of 143 ± 7.77 at day 1 to 155 ± 11.17 at 21st day. This method gave concordant analysis as given for yeast biomass concentration where cell counting is not possible (Acevedo et al., 2009). However, this was just a small scale check and the same could be done using bigger air pillows of LDPE plastic wrap material whereby the main aim was to keep the system closed, have enough permeability for CO2 and other gases while simultaneously preventing water loss, avoiding contamination and frequent inputs of nutrient media.