Three SLFs had abundant nutrients that are required for microalgal growth. Macro and trace nutrients were found to be high in both red seaweed media (SLF-1, SLF-2) in comparison with the brown seaweed media (SLF-3). The essential macronutrients N and K were high in SLF-1 whereas P content was high in SLF-2. An important trace nutrient that plays a major role in microalgal growth is iron which was found to be more in SLF-1 followed by SLF-2 whereas low in SLF-3. Among the macro nutrients, sodium and potassium was most abundant followed by calcium and nitrogen. In the study conducted by Zheng et al. (2016), the kelp waste extract contained enormous amount of nitrogen (5723 mg/L), phosphorous (5529 mg/L) and potassium (60.54 mg/L) whereas iron was present at not detectable levels. The results of the nutrient analysis suggest that the SLFs prepared from the seaweeds were loaded with essential nutrients for microalgal growth. This media can act as a biostimulant that can satisfy the nutrient requirement of microalgae for growth as well as lipid production. Villares et al. (2007) has reported that seaweeds contain plenty of macro, micro and trace nutrient elements. Biomass and lipid productivity of the microalgae depends on the nutrients available in the culture media. Nutrient elements present in seaweed extract were found to be water soluble in nature hence get easily absorbed by plants and helps in reducing the nutrient deficiency problems (Mohanty et al. 2013). The results of the present study suggest that high levels of nutrients were present in the SLFs prepared from red seaweed in comparison with brown seaweed media. These results are in agreement with the report of Raja et al. (2015) that the extract of the red seaweed K. alvarezii had higher amount of nutrients than the brown seaweed T. cornoides extract. Thus, it can be concluded that, the red seaweed could be effectively utilized as an organic source for the media preparation for the growth and maintenance of the marine microalgae.
SLFs had a strong stimulation on the growth of both microalgae. The results of the growth study suggests that SLF-2 as the best culture medium followed by SLF-1 which enhances the biomass production of N. oculata to a greater level in comparison with SLF-3 as well as the control. The best media dose for enhancing the biomass production using SLF-1, SLF-2 and SLF-3 were 6, 8 and 2% respectively. Two red seaweed SLFs resulted in higher biomass production in comparison with the commercial media suggesting the efficiency of the seaweed as an effective microalgal culture media. Similarly, enhancement in the growth of microalgae using Kelp waste extract was reported by Zheng et al. (2016) that Chlorella sp., C. sorokiniana, S. maxima and P. tricornutum exhibited a higher growth in KWE than the control (commercial media). Studies on seaweed extract added with commercial media also enhanced the biomass production. A study on the effect of red seaweed extracts of Gracilaria corticata and Grateloupia lithophila added as supplement with the commercial BBM media resulted in an increase of cell density of Chlorella vulgaris than the BBM exclusively grown microalgae (Lakshmi and Sheeja, 2021).
The halophilic dinoflagellate D. salina grown under three different SLFs suggest that the best medium to obtain maximum biomass production was SLF-1 followed by SLF-2. Both the red seaweed media resulted in higher biomass production than the commercial medium grown microalgae. SLF-3 showed a significantly lower cell density than the control. The result of the present study suggests the best concentrations of SLF-1, SLF-2 and SLF-3 for achieving maximum biomass production as 6, 10 and 4% respectively. SLF-1 resulted in higher biomass value even at low medium concentration (6%) than the SLF-2 (10%). These results well corelate with the findings of Rohani-Ghadikolaei et al. (2012) that the seaweed extracts (U. lactuca, E. intestinalis, G. corticate) used as supplement along with the commercial F/2 medium resulted in enhancement of Isochrysis galbana growth in comparison with those achieved by commercial media exclusively. Another report by Cho et al. (1998) also confirms that there is enhancement in growth of microalga Isochrysis galbana when grown in the extract of seaweed Monostroma nitidum. Crouch and Van Staden (1993) stated the reason behind the enhancement of microalgal growth by seaweed extract (SWEs), that SWEs are not only abundant with supplementary nutrients but also with the growth enhancing substances such as auxins and cytokinins, which results in the enhancement of the growth of microalgae.
In the present study, both the microalgae showed significantly lower growth in SLF-3 treatment. The two main reason behind the low growth exhibited by both the microalgae grown in brown SLF may be due to the low nitrogen and iron content as well as the dark colour of the media. The presence of low nutrient content may cause nutrient deficiency thereby declining the microalgal growth whereas the darkness of the media may reduce the penetration of light thereby inhibiting the microalgal growth. It was also observed that low concentration of the SLF-3 supported growth to an extent whereas the high concentration inhibited growth in higher level. Similar result was also noted in the study of Malik et al. (2018) that the growth of Chaetoceros gracilis was reduced when grown at media added with higher concentration of seaweed extract of Sargassum sp. Another documentation by Alvarado et al. (2008) states that Chaetoceros muelleri grown in the brown seaweed macroalgal compost supplemented with di-silicate resulted in less growth in comparison with the microalgae grown in Walne’s medium.
The maximum cell density of microalgae N. oculata grown under SLF-1, SLF-2 and SLF-3 was achieved on the 12th, 14th and 14th day respectively suggesting that SLF-1 has shorten the life cycle, whereas for D. salina maximum growth was achieved on the 12th, 10th and 14th day respectively showing that SLF-2 medium had reduced the life cycle of the microalgae. These results are in agreement with report of Zheng et al. (2016) that the organic media had shorten the life cycle of microalgae grown in KWE. The best medium to obtain the maximum microalgal production of N. oculata was SLF-2 at 8% concentration whereas for D. salina the medium was SLF-1 at 6% concentration. Different microalgal species reacts differently when exposed with different organic media of different doses. This varies with microalgal species based on their level of tolerance level to the exposed organic compounds (Zhang et al. 2014).
The biochemical composition of the microalgae can be well correlated with the nutritional quality of the culture medium in which it was cultivated (Zhang, 1997). Selection of suitable culture media mainly focuses on the biomass production at the same time the biochemical composition shouldn’t get affected. The present study concludes that the best organic medium for N. oculata to achieve the maximum protein content was SLF-2 medium followed by SLF-1 whereas for D. salina the best medium was SLF-1 followed by SLF-2. Both the microalgae showed low protein content when grown under SLF-3 suggesting the red seaweed media as the suitable and effective culture media. The present study emphasizes that the protein content of the microalgae was found to be increasing when grown under the SLFs in comparison with control. Similar results of increase in microalgal protein content by seaweed extracts was mentioned by Rohani-Ghadikolaei et al. (2012) that when seaweed extracts of Gracilaria corticate, Ulva lactuca and Enteromorpha intestinalis were added as supplement with the commercial media F/2, it resulted an increase of protein content of the microalgae than grown exclusively with F/2 media. Another report documents that there was a slight enhancement of protein concentration of the microalgae grown under BBM media amended with red seaweed extract of Gracilaria corticata and Grateloupia lithophila (Lakshmi and Sheeja, 2021). The decrease in protein content of microlage grown using brown seaweed medium was reported by Alvarado et al. (2008) that when Chaetoceros muelleri was grown under brown seaweed compost added with silicate it resulted lower protein content of 2–5% whereas in control the protein value was 12%. Earlier reports and present study results concludes that red seaweed media enhances the protein value of the microalgae whereas the brown seaweed decreases it significantly.
SLFs has enhanced the pigment synthesis in microalgae which resulted in higher chlorophyll and carotenoid production than the commercial F/2 medium. Both the red seaweed media ie., SLF-1 and SLF-2 resulted in higher chl-a production in N. oculata that was 1.2 and 1.22 times higher than the control respectively whereas in SLF-3 treatment the chl-a content was 1.26 times lower than the control. Similarly, carotenoid production was also enhanced to 1.04 and 1.19 times higher by SLF-1 and SLF-2 than by the commercial media. In the halophyte D. salina, chl-a, b and carotenoid production were significantly enhanced by the red seaweed SLFs. SLF-1 and SLF-2 resulted in 2 and 1.76 times higher chl-a production; 1.42 and 1.33 times higher chl-b production; 1.85 and 2.01times higher carotenoid production than the control respectively. SLF-3 has decreased the pigment synthesis ie., chl-a, b and carotenoid content by 1.03, 134 and 1.14 times than the control respectively. In comparing the efficiency of both red seaweed SLFs, SLF-2 enhanced the pigment synthesis of N. oculata in a higher level than the SLF-1. In D. salina, SLF-1 enhanced chl-a and b level higher than the SLF-2 but carotenoid production was more enhanced by the SLF-2 when compared to SLF-1. Enhancement of the pigment synthesis in microalgae Chlorella vulgaris was noted when the seaweed extracts of Gracilaria corticata and Grateloupia lithophila were added as supplements in the commercial media BBM (Lakshmi and Sheeja, 2021). The extracts of Kappaphycus alvarezii and Turbinaria conoides added as supplement with F/2 and Walne’s media for culture of C. muelleri and D. salina resulted in increase of pigment synthesis (chl-a and b production) in both the microalgae (Raja et al. 2015). Our results and the previous reports also conclude that SWE has the potential to increase the pigment synthesis in microalgae. The reason behind this potential may be due to the presence of high level of phytohormones like cytokinins in the SWE that will increase the nutrient absorption and utilization capacity of microalgae and thereby increasing the pigment production (Zhang, 1997). The present study also suggest that the brown seaweed medium has reduced the pigment synthesis of microalgae, the main reason for this may be due to the low content of iron available in the media which remains a main key factor in deciding the pigment synthesis potential of microalgae (Kong et al. 2014).
The red seaweed SLFs had a positive influence on the lipid synthesis of both microalgae. SLF-1 and SLF-2 had enhanced the lipid production of N. oculata by 1.19 and 1.13 times higher than the control whereas SLF-3 reduced the lipid synthesis by 1.3 times. In D. salina, the lipid production was increased by SLF-1 and SLF-2 to 1.4 and 1.6 times than the control and decreased by 1.42 times by SLF-3. In N. oculata, the lipid production was greatly enhanced by SLF-1 whereas in D. salina, lipid synthesis was highly increased by SLF-2. SLF-3 has reduced the lipid synthesis in both the marine microalgae. These results are comparable with the results of Alvarado et al. (2008) that there was an increase in lipid production of microalgae C. muelleri when grown in seaweed compost amended with di-sodium silicate. The lipid production in microalgae cultured in seaweed compost media was 42% whereas in control (cultured in Walne medium) it was only 28%. Another study documented by Gireesh51 is also in agreement with the present study results, that there was a notable increase in lipid production of D. salina grown in seaweed liquid fertilizer than the microalgae cultured in commercial media (Conveyor Walne’s media). Rohani-Ghadikolaei et al. (2012) has also given similar results of enhanced microalgal lipid production in microalgae I. galbana when cultivated in seaweed extracts as a supplement with the commercial media (f/2 medium). The potentiality of seaweed extracts in enhancing the lipid synthesis in microalgae could be due to the presence of abundant levels of carbon in seaweed extracts which are in convertible form that can stimulate the lipid synthesis and accumulation in microalgae (Liang et al. 2009).
EPA content of N. oculata grown using SLF 1 and 2 was increased by 19.85% and 6.35% respectively. In D. salina, methyl palmitoleate concentration was increased up to 21.46% when grown using SLFs as culture media. In both the microalgae using commercial media, DHA was not detected whereas it was found at trace levels in both red SLFs treatment. The fatty acid study results reveals the efficiency of SLFs in increasing the composition of fatty acids and its concentration. Both the SLFs prepared from red seaweeds had enhanced the EPA and palmitoleic acid content of N. oculata and D. salina respectively suggesting its potential application in aquaculture sectors. There are no studies reported on the effect of seaweeds as liquid fertilizer for the growth of microalgae. But the study by Zheng et al. (2016) on the effect of kelp waste extract on microalgae suggest that it has reduced the PUFA synthesis of Chlorella strains whereas increased the synthesis of SFA.
There are few reports stating the effect of seaweed extracts as microalgal culture media that were added as supplement with the commercial media. This study provides an eco-friendly and completely alternative culture medium that has a shelf life of more than one year. This report shall be considered as the first study that documents an effective and cheap cost culture media for microalgae with a longer shelf life. The organic fertilizer reported by Lam and Lee (2012), used for microalgal growth stimulation cost around 1.20 USD/400 g whereas another study by Zheng et al. (2016) documented the cost of production of one litre Kelp Waste Extract (culture media for microalgae) cost around 0.28 USD. The present study documents the overall production cost of one litre of SLF with an estimate of 0.1 USD (10 g cellulase cost) by the enzymatic hydrolysis method, that is very much lower than the production cost of previously reported organic media and commercial media. Keeping this in view, it can be practically applicable in aquaculture and biofuel industries.