Sustainable Fish Feeds: Optimization of Levels of Inorganic Fertilizers for Mass Production of Oocystis Sp. For Climate Smart Aquaculture

Sustainable Fish Feeds: Optimization of Levels of Inorganic Fertilizers for Mass Production of Oocystis Sp. 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For Climate Smart Aquaculture Marvin Gaye Mukoma, Steve Omondi Odour, Elick Onyango Otachi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1637529/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2022 Read the published version in Aquaculture International → Version 1 posted 9 You are reading this latest preprint version Abstract Use of microalgae as source of food in aquaculture production is gaining recognition due to their rapid growth rate that promises high biomass generation within a short time. The challenge faced is getting good and inexpensive nutrients source to be used in mass production of the required microalgae. This study investigated the effect of different nutrient combination in influencing the growth rate of the of the green algae Oocystis sp. which has been indentified as possible protein source for the raising of Orechromis niloticus fingerlings for fish farming. Modified Bolds 3N Medium and commercial agricultural fertilisers (urea, NPK and DAP) media were compared to establish the appropriate combination that would result into high biomass generation but at the lowest cost possible. The Modified Bold 3N Medium acted as the control, at a cost of 11.28 KSh per litre, the other media were derived from urea, NPK and DAP (varying the ratio of each) at a cost of treatment 1 (0.14 KSh per litre), treatment 2 (0.18 KSh per litre) and treatment 3 (0.22 KSh per litre). The algae was cultured for five weeks with samples taken daily for biomass analyses using chloropyhll-a concentration as the surrogate for Oocystis sp. biomass for 30 days, from each treatment was determined. The growth rate, doubling time, and divisions per day were then estimated based on this chlorophyll-a concentration. The results showed that the mean concentrations of chlorophyll-a in treatment 1 was highest (7.715 ± 0.667 µg/ml) while treatment 3 (6.441 ± 0.555 µg/ml) had the least. There was no significant differences in the mean concentrations of chlorophyll-a in the four treatments (Kruskal-Wallis H test: P > 0.05). The chlorophyll-a concentration varied significantly in each treatment with time (Kruskal-Wallis H test: P 0.05), divisions per day (Kruskal-Wallis H test: P > 0.05), and doubling time (Kruskal-Wallis H test: P > 0.05) from the different treatments. The results of this study showed that inorganic fertilizers can be used as cost-effective media in the mass scale culture of Oocystis sp. Oocystis sp. Mass production Inorganic fertilizers Climate smart Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Microalgae are associated with diverse applications such as biodiesel production and animal feed sources due to their faster growth rates that yield high biomass within a short time (Mata et al. 2010 ; Khan et al. 2018 ). Some species of microalgae have high lipid accumulation in their dry cell weight ranging between 50–60% that makes them good candidates for use as energy sources (Hu et al. 2008 ; Mata et al. 2010 ). Saadaoui et al. ( 2021 ) observed that some microalgae have been used as food in livestock, poultry and aquaculture production due to their diverse nutritional properties. For example Chlorella vulgaris was used as a substitute for fish meal in feed for Clarias gariepinus (Enyidi 2017 ). In another study, El-Sheekh et al. ( 2014 ) used Arthrospira platensis as feed for hybrid red tilapia ( Oreochromis niloticus x Oreochromis mossambicus ). These studies revealed that the inclusion of algae in the feeds was beneficial to the cultured fish through an increase in the feed conversion ratio. Some other species of such as Oocystis sp. has been reported to have varying amounts of proteins in the cells, ranging between 15 to 40% ( Lee and Picard 1982 ). The differences in the protein contents have been attributed to the culture medium. However, Oocystis sp., incorporation in fish feeds has not yet been done investigated comprehensively. The expansion of microalgae production is critical in any perceived applications, hence the need for development of cost-effective media for microalgae cultivation. The factors that affect microalgae growth are namely light, temperature, and nutrients (da Silva Ferreira and Sant'Anna 2017; Gani et al. 2019 ; Kazbar et al. 2019 ; Chowdury et al. 2020 ). For their nutrient requirements this varies with each algal species having a specific requirement to optimize growth (Ghafari et al. 2018 ; Khan et al. 2018 ). Nitrogen and Phosphorus are essential as they are limiting factors to microalgae growth (Yaakob et al. 2021 ). Consequently, Nitrogen and Phosphorus sources are usually emphasized while upscaling the culture of microalgae. Raising interest in the use of inorganic fertilizers in microalgae culture, as a source of nutrients; which stands out as a major limitation in large-scale production of algae (Hu et al. 2008 ; Mata et al. 2010 ; Ravindran et al. 2016 ) is critical. Various studies have been done assessing the use of inorganic fertilizers (either singly or combined) in microalgae culture (Ashraf et al. 2011 ; Nayak et al. 2016 ; Michael et al. 2019 ). The inorganic fertilizers commonly used in mass-scale production are urea, Di-ammonium Phosphate (DAP), and NPK (Ansari et al. 2017 ; Arenas et al. 2017 ; Win et al. 2018 ; Renuka et al. 2018 ). Urea is a nitrogenous fertilizer that consists of 46% of nitrogen (having the highest nitrogen content). DAP is important as it provides both nitrogen and phosphorus for growth, found having different ratios depending on the fertilizer's end-use. NPK is also an inorganic fertilizer that is wholesome as it provides nitrogen, phosphorus, and potassium nutrients for the growth of organisms, with individual concentrations differing based on the end-use. Different concentrations of the inorganic fertilizers affect the growth of microalgae; further, a combination of different fertilizers is used to provide microalgae with all the nutrients required for growth. The aim of this study was to establish the optimum level of inorganic fertilizers combination that could be used in the mass production of Oocystis sp. to give maximum yield, by focusing on urea level inclusion, with the ultimate goal of upscaling microalgae culture using cost-effective media. Materials And Methods 1.3.1 Study area The study was conducted at Egerton University in the Biological Sciences Department laboratories. The University is located in Nakuru County, Njoro Sub- County, and is approximately 25 km south-west of Nakuru city, at an altitude of 1890–2190 m above sea level. The area's temperature ranges between 17 0 C -27 0 C (Waithaka et al. 2017 ). 1.3.2 Culturing of Oocystis sp. The algae was cultured using Modified Bolds 3N Medium and commercial agricultural fertilizers (urea, NPK and DAP) media for a period of five weeks. The inoculum for culturing Oocystis sp. was obtained from the University of Texas Culture Collection of Algae(UTEX). Modified Bolds 3N Medium (UTEX, n.d.) was used as a control. The other treatments were as shown in Table 1 . Pure cultures were raised through isolation and growing the culture in the incubators in the laboratory to raise suitable biomass before transferring them into 3-liter plastic bottles as growth chambers or bioreactors containing the culture media. Table 1 Composition of different treatment culture media. Treatments NPK (g/L) UREA (g/L) DAP (g/L) Treatment 1 0.75 0.25 0.25 Treatment 2 0.75 0.5 0.25 Treatment 3 0.75 0.75 0.25 1.3.3 Sampling and biomass estimation Samples were taken daily after 24 hours for 30 days, where 20ml was filtered through Glass Fibre Carbon filters from each treatment bottle. Chlorophyl-a concetration was determined using the standard method as given in the American Public Health Association (APHA) ( 2005 ). Extraction of the chlorophyll-a was done using acetone, where the filters and seston were folded and covered by aluminium foil and stored in a freezer overnight to aid in the bursting of the cells. The seston and the filters were then homogenised in a tissue grinder (Heidolph, 637 69, Germany) at around 5,000-rpm where 5 ml of 90% aqueous acetone was added. The samples were then transferred into a centrifuge tube, the grinder rinsed with 90% acetone (volume used was noted), and the rinsed slurry was added to the extraction slurry. The volume was then adjusted to 10 ml with 90% acetone and left for at least 8 hrs in the dark at 4°C for full chlorophyll-a extraction. After incubation, the samples were centrifuged for 10 min at 3,000-rpm. The clarified extract was decanted into a clean test tube. Light absorbance of the chlorophyll-a extract was measured with a spectrophotometer (Pharmacia Biotech Novaspec II, Sweden) at 750 nm and 663 nm. Chlorophyll-a concentration was calculated using, the following equation according to Talling & Driver ( 1963 ): Biomass; \(N=\frac{11.4*\left(B-A\right)*Ve}{Vf*Lp}\) 1 Where; B = absorbance at 663 nm A = absorbance at 750 nm Ve = volume of extract used (ml) Vf = volume of sample filtered (ml) Lp = light path length of the cuvette (cm) After which the Chlorophyll-a concentration was used to derive the growth rate, divisions per day, and the generation time as critical parameters of importance. These were all derived from the equations developed by Levasseur et al. ( 1993 ): Growth rate; \({K}^{\text{'}}=\frac{Ln\left(\frac{N2}{N1}\right)}{t2-t1}\) 2 Where N1 and N2 = biomass at time1 (t1) and time2 (t2) respectively; Divisions per day and the doubling time were calculated based on the specific growth rate. Divisions per day; \(\frac{Div}{day}=\frac{{K}^{\text{'}}}{Ln2}\) 3 Where Ln2 = Natural logarithm to the base 2 Doubling time; \(Doub\text{'} t =\frac{1}{\frac{Div}{day}}\) 4 1.3.4 Statistical analyses The biomass, growth rate, divisions per day, and doubling time of Oocystis sp. cultured in the different treatments were compared using Sigma Plot software (version 14) through Kruskal-Wallis H test, as the data failed Normality Test (Shapiro-Wilk: P < 0.050). Results 1.4.1 Water quality Mean temperature during the study ranged from 24.23 0 C to 27.33 0 C. Temperature variations during the study period were not significant among the treatments (Kruskal-Wallis H test: P > 0.05). The pH for Treatment 1 ranged from 6.8 to 7.7, where there was a steady increase from the beginning of the study. On the other hand, pH for Treatment 2 ranged from 6.6 to 7.4, and Treatment 3 ranged from 6.4 to 7.2, all following a similar trend as that of Treatment 1 characterised by an increase in the pH. Finally, the control treatment had a pH ranging from 6.1 to 7.1, also having a similar trend in time with the other three treatments 1.4.2 Comparison of chlorophyll-a concentration at different fertilizer treatments. The concentrations o of chlorophyl-a in Treatment 1 was highest (7.715 ± 0.667 µg/ml) followed by the Control (6.963 ± 0.788 µg/ml) and Treatment 2(6.862 ± 0.617 µg/ml), with the least in Treatment 3 (6.441 ± 0.555 µg/ml). There were no statistically significant differences in mean concentrations of chlorophyll-a among different treatments (Kruskal-Wallis H test: P > 0.05). 1.4.3 Temporal variations in chlorophyll-a concentration. Generally, the concentration increased gradually in all treatments until it reached its peak on different days for all the treatments. The control treatment (Modified Bolds 3N Medium) achieved the highest chlorophyll-a concentration on the 25th day (12.977 ± 0.788 µg/ml). Treatment 1 followed, reaching its highest chlorophyll-a concentration on the 17th day (11.4437 ± 0.667 µg/ml). Treatment 2 achieved its highest concentration on the 21st day (11.3696 ± 0.617 µg/ml), and finally Treatment 3 had the least concentration, which was achieved on the 16th day (10.2714 ± 0.555 µg/ml). Overall, the chlorophyll-a concentration in all the treatments fluctuated once the optimum concentration was achieved. At the end of the experiment, the concentration of chlorophyll-a in the treatments followed a similar pattern, with the control having the highest (11.2442 ± 0.788 µg/ml) while treatment 3 had the least (5.8197 ± 0.555 µg/ml) (see Figs. 1 to 4 ) The chlorophyll-a concentration varied significantly in each treatment with time (Kruskal-Wallis H test: P < 0.001) for all the treatments. At the beginning of the study, all the treatments had equal chlorophyll-a concentration (where 100 ml of stock Oocystis sp. cultured having a concentration of 10.0719 µg/ml was introduced into 2.49 litres of respective treatment media). 1.4.4 Growth rate, Divisions per day, and Doubling time The maximum, mean, and least values of the growth rate, divisions per day, and doubling time in different treatments are shown in Table 2 . There was no significant difference in the mean growth rate(Kruskal-Wallis H test: P > 0.05), divisions per day (Kruskal-Wallis H test: P > 0.05), and doubling time (Kruskal-Wallis H test: P > 0.05) for the treatments. Table 2 The maximum, mean, and least values of the growth rate, divisions per day, and doubling time of different treatment culture media. Parameters Treatment Control Treatment 1 Treatment2 Treatment 3 Growth rate max 0.376 0.601 0.469 0.545 Growth rate mean 0.109 0.0963 0.0885 0.0837 Growth rate least -0.215 -0.209 -0.219 -0.213 Divisions per day max 0.543 0.867 0.676 0.786 Divisions per day mean 0.157 0.139 0.128 0.121 Divisions per day least -0.310 -0.302 -0.316 -0.307 Doubling time max 1028.630 70.142 187.374 37.900 Doubling time mean 49.722 -11.267 13.352 1.412 Doubling time least -124.118 -532.684 -9.929 -39.418 1.4.5 Media Cost and Biomass The control (Modified Bold 3N Medium) was produced at a cost of 11.28 KSh per litre, the other media were derived from urea, NPK and DAP at a cost of 0.14 KSh per litre for treatment 1, 0.18 KSh per litre for treatment 2 and 0.22 KSh per litre for treatment 3. Distilled water was generated by a distiller in the laboratory for use in generating the control media (Modified Bold 3N Medium) while harvested rain water was used for the experiment for treatment 1,2 and 3; hence, their cost were not captured in the above calculation. Discussion The nature of the culture media is the main determining factor in the growth of microalgae, their productivity and ultimately their biomass as long as the pH, light intensity, and temperature needs have been fulfilled (da Silva Ferreira and Sant'Anna, 2017; Gani et al. 2019 ; Chowdury et al. 2020 ). The light provided to the culture was by white light-emitting diode tubes (3200K, 9W). Csavina et al. ( 2011 ) illustrated that Oocystis sp. requires the optimum light intensity of 150 µ mol m − 2 s − 1 . Latala et al. ( 1991 ) found that the growth of Oocystis sp. was completely inhibited at high light intensities ranging between 270–380 µE/m 2 s. pH influences the quantity of free carbon in the culture media and the balance between carbonate and bicarbonate. In this study, the pH was adjusted to range between 6.0 to 8.0, as reported by RAo ( 1963 ), to suit the growth of Oocystis sp. optimally. Mayo ( 1997 ) found similar results while culturing Chlorella sp. with the maximum growth rate at pH ranging from 6.31 to 6.84. Changes in the media pH were attributed to the photosynthetic and decomposition process of the cultured Oocystis sp. ( Tucker and D'Abramo 2008; Rahardini et al. 2018 ). During the study, the water temperature range was in optimal range as reported by Nalley et al. ( 2018 ), where they found the best temperature for optimum growth of Oocystis sp. ranged between 26.60 0 C to 27.78 0 C. Chowdury et al. ( 2020 ) state that temperature is critical in micro-algae culture as it affects the algal growth rate, cell size, biochemical composition, and nutrient requirements. The temporal chlorophyll-a concentration varied amongst the treatments, mainly due to the different constituents in the culture treatment media, with the differences being significant with time in all the treatments. Sutkowy et al. ( 2019 ) state that the composition of culture media influences the growth of micro-algae in vitro. Overall, all treatments followed a similar trend where there was a decrease in the chlorophyll-a concentration once it was at its highest biomass. The pattern achieved is similar to that obtained by Rahardini et al. ( 2018 ) while assessing the growth of Chlorella sp., which is also found in the Chlorophyceae class of Green algae. At the begging of the experiment, there was a steady increase (exponential phase) in the chlorophyll-a concentration due to sufficient nutrient concentration in all the treatments. There was no lag phase observed during the study, which can be attributed to the conditions of the inoculum (Spencer 1954 ). Talling ( 1966 ) notes that an inoculum obtained from a healthy exponentially growing population is unlikely to have a lag phase when transferred to a freshly prepared media under similar growth conditions (light, temperature, and pH); consequently, there is no need for physiological adaptations for growth. The lack of a lag phase is crucial as it reduces the time required for upscaling the culture, allowing the harvesting of cultured cells sooner. The exponential phase is characterised by the production of materials that are also capable of growth (Fogg and Thake 1987 ), with its length depending on the size of the inoculum, the growth rate, and the capacity of the medium and culturing conditions to support algal growth. The exponential phase was followed by the stationary phase, characterised by a decline in population growth, resulting from reduced nutrient concentration in the culture media, causing the number of cells to remain constant. The stationary phase is characterised by zero net growth, as the rate of the growth of the cells is equal to the rate of cell death ( Nyström 2004 ; Maier and Pepper 2015 ). Finally, the death phase follows, as nutrients in the culture media run out, resulting in the death of cultured algal cells; therefore, a decline in the chlorophyll-a concentration was witnessed. The death rate of cells in the media became higher than the rate of cell growth (Maier and Pepper 2015 ). The study demonstrated that the inorganic fertilizers' specific compositions and comparison between the culture media did not have any significant impact on the growth rate, divisions per day, and the doubling time of cultured Oocystis sp.. Similar results were also obtained by Rahardini et al. ( 2018 ) who also found no significant differences in the growth rate and doubling time of Chlorella sp. cultured in media with different compositions. The divisions per day obtained was higher than those from Csavina et al. ( 2011 ), which can be attributed to the continuous agitation of the algal cells during culture, consequently not allowing the clamping of cells together, resulting in deaths and ultimately decline in the growth rate and divisions per day. Research carried out by Sobczuk et al. ( 2006 ) showed an increase in the growth rate of microalgae with an increase with agitation; it should also be noted that excessive agitation can result in cell death. Conclusion This study found that inorganic fertilizers can be used as an option of cost-effective media in the culture of Oocystis sp., with the ratio of 3:1:1 while using NPK, urea, and DAP fertilizers respectively to achieve the highest biomass within the shortest period. The finding that there were no significant differences in the Oocystis sp. biomass using either medium is an encouraging result as it offers opportunities to explore the use of low-cost media which is equally competitive with the standard media in mass production of Oocystis sp. for potential use in fish feeds in Aquaculture. Declarations Funding This work was supported by The Kenya Climate Smart Agriculture Project (KCSAP) Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mukoma, Marvin Gaye. 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Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2022 Read the published version in Aquaculture International → Version 1 posted Editorial decision: Accepted 20 Sep, 2022 Reviews received at journal 04 Aug, 2022 Reviewers agreed at journal 23 Jul, 2022 Reviews received at journal 18 Jun, 2022 Reviewers agreed at journal 17 Jun, 2022 Reviewers invited by journal 17 May, 2022 Editor assigned by journal 13 May, 2022 Submission checks completed at journal 10 May, 2022 First submitted to journal 09 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1637529","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":104881010,"identity":"cef62417-941f-42d8-a6c8-d5041741d023","order_by":0,"name":"Marvin Gaye Mukoma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYLCCBCCWADE+ADEbOylaGGeAtDATaxNICzMPiEVIi2772WcSD3dsS5zZ3p342ebXNnk+ZgbGDx9zcGsxO5NuJpF45nbibJ6zm6Vz+24btjEzMEvO3IZHy4E0ZoPEttuJ8yRyN0jn9txmBGphY+bFp+X8M7iWzb8te27bE9ZyI43xAUjLbIncbdIMP24nEqHlGViL8cyes9ssextuJ7cxMzbj98v5NIaDP9tuy8443rv5xo8/t23ntzcf/PARjxZUwNgGJhuIVQ8Cf0hRPApGwSgYBSMFAABNyFVHybsQcAAAAABJRU5ErkJggg==","orcid":"","institution":"Egerton University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Marvin","middleName":"Gaye","lastName":"Mukoma","suffix":""},{"id":104881012,"identity":"c7e56f60-aa9e-4878-afdb-d21982859a91","order_by":1,"name":"Steve Omondi Odour","email":"","orcid":"","institution":"Egerton University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Steve","middleName":"Omondi","lastName":"Odour","suffix":""},{"id":104881014,"identity":"5de660d5-92e9-40b8-bb30-207808e68128","order_by":2,"name":"Elick Onyango Otachi","email":"","orcid":"","institution":"Egerton University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elick","middleName":"Onyango","lastName":"Otachi","suffix":""}],"badges":[],"createdAt":"2022-05-09 09:14:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1637529/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1637529/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10499-022-00986-5","type":"published","date":"2022-10-01T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":21514054,"identity":"6a091758-0bf9-4c6b-88c9-7a5f4fc865cd","added_by":"auto","created_at":"2022-05-16 15:11:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":182143,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal variations in chlorophyll-a concentration in the Control treatment\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1637529/v1/d46f643195a3a4a79c5e92be.png"},{"id":21514053,"identity":"b59e0762-e45b-4e67-b88e-df52353e7edc","added_by":"auto","created_at":"2022-05-16 15:11:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":167963,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal variations in chlorophyll-a concentration in the Treatment 1\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1637529/v1/e9b2752ce0dcea92a4f47bdd.png"},{"id":21514052,"identity":"4082e00d-f9e0-47b4-b92a-f5b3ccb24a51","added_by":"auto","created_at":"2022-05-16 15:11:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177723,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal variations in chlorophyll-a concentration in the Treatment 2\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1637529/v1/5950081fe247d9d7efc45cc4.png"},{"id":21514055,"identity":"d0ae31e5-a147-4400-834a-b828ab3fdbd8","added_by":"auto","created_at":"2022-05-16 15:11:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":170919,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal variations in chlorophyll-a concentration in the Treatment 3\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1637529/v1/e586b3a89e8940b8ccf64bbb.png"},{"id":33363353,"identity":"04dd1e35-691b-4d51-8aff-2a081936805f","added_by":"auto","created_at":"2023-02-23 18:33:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":675684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1637529/v1/ae440cf7-b70b-4301-b963-b8b68bdb13df.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSustainable Fish Feeds: Optimization of Levels of Inorganic Fertilizers for Mass Production of Oocystis Sp. For Climate Smart Aquaculture\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicroalgae are associated with diverse applications such as biodiesel production and animal feed sources due to their faster growth rates that yield high biomass within a short time (Mata et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Khan et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Some species of microalgae have high lipid accumulation in their dry cell weight ranging between 50\u0026ndash;60% that makes them good candidates for use as energy sources (Hu et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mata et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eSaadaoui et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) observed that some microalgae have been used as food in livestock, poultry and aquaculture production due to their diverse nutritional properties. For example \u003cem\u003eChlorella vulgaris\u003c/em\u003e was used as a substitute for fish meal in feed for \u003cem\u003eClarias gariepinus\u003c/em\u003e (Enyidi \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). In another study, El-Sheekh et al. (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) used \u003cem\u003eArthrospira platensis\u003c/em\u003e as feed for hybrid red tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e x \u003cem\u003eOreochromis mossambicus\u003c/em\u003e). These studies revealed that the inclusion of algae in the feeds was beneficial to the cultured fish through an increase in the feed conversion ratio.\u003c/p\u003e\n\u003cp\u003eSome other species of such as \u003cem\u003eOocystis\u003c/em\u003e sp. has been reported to have varying amounts of proteins in the cells, ranging between 15 to 40% ( Lee and Picard \u003cspan class=\"CitationRef\"\u003e1982\u003c/span\u003e). The differences in the protein contents have been attributed to the culture medium. However, \u003cem\u003eOocystis\u003c/em\u003e sp., incorporation in fish feeds has not yet been done investigated comprehensively.\u003c/p\u003e\n\u003cp\u003eThe expansion of microalgae production is critical in any perceived applications, hence the need for development of cost-effective media for microalgae cultivation. The factors that affect microalgae growth are namely light, temperature, and nutrients (da Silva Ferreira and Sant\u0026apos;Anna 2017; Gani et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kazbar et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chowdury et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). For their nutrient requirements this varies with each algal species having a specific requirement to optimize growth (Ghafari et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Khan et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nitrogen and Phosphorus are essential as they are limiting factors to microalgae growth (Yaakob et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, Nitrogen and Phosphorus sources are usually emphasized while upscaling the culture of microalgae.\u003c/p\u003e\n\u003cp\u003eRaising interest in the use of inorganic fertilizers in microalgae culture, as a source of nutrients; which stands out as a major limitation in large-scale production of algae (Hu et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mata et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ravindran et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) is critical. Various studies have been done assessing the use of inorganic fertilizers (either singly or combined) in microalgae culture (Ashraf et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e; Nayak et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Michael et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe inorganic fertilizers commonly used in mass-scale production are urea, Di-ammonium Phosphate (DAP), and NPK (Ansari et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Arenas et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Win et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Renuka et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Urea is a nitrogenous fertilizer that consists of 46% of nitrogen (having the highest nitrogen content). DAP is important as it provides both nitrogen and phosphorus for growth, found having different ratios depending on the fertilizer\u0026apos;s end-use. NPK is also an inorganic fertilizer that is wholesome as it provides nitrogen, phosphorus, and potassium nutrients for the growth of organisms, with individual concentrations differing based on the end-use. Different concentrations of the inorganic fertilizers affect the growth of microalgae; further, a combination of different fertilizers is used to provide microalgae with all the nutrients required for growth.\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to establish the optimum level of inorganic fertilizers combination that could be used in the mass production of \u003cem\u003eOocystis\u003c/em\u003e sp. to give maximum yield, by focusing on urea level inclusion, with the ultimate goal of upscaling microalgae culture using cost-effective media.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section3\" id=\"Sec3\"\u003e\n \u003ch2\u003e1.3.1 Study area\u003c/h2\u003e\n \u003cp\u003eThe study was conducted at Egerton University in the Biological Sciences Department laboratories. The University is located in Nakuru County, Njoro Sub- County, and is approximately 25 km south-west of Nakuru city, at an altitude of 1890\u0026ndash;2190 m above sea level. The area\u0026apos;s temperature ranges between 17\u003csup\u003e0\u003c/sup\u003eC -27\u003csup\u003e0\u003c/sup\u003eC (Waithaka et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section3\" id=\"Sec4\"\u003e\n \u003ch2\u003e1.3.2 Culturing of \u003cem\u003eOocystis\u003c/em\u003e sp.\u003c/h2\u003e\n \u003cp\u003eThe algae was cultured using Modified Bolds 3N Medium and commercial agricultural fertilizers (urea, NPK and DAP) media for a period of five weeks. The inoculum for culturing \u003cem\u003eOocystis\u003c/em\u003e sp. was obtained from the University of Texas Culture Collection of Algae(UTEX). Modified Bolds 3N Medium (UTEX, n.d.) was used as a control. The other treatments were as shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Pure cultures were raised through isolation and growing the culture in the incubators in the laboratory to raise suitable biomass before transferring them into 3-liter plastic bottles as growth chambers or bioreactors containing the culture media.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComposition of different treatment culture media.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNPK (g/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUREA (g/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDAP (g/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTreatment 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section3\" id=\"Sec5\"\u003e\n \u003ch2\u003e1.3.3 Sampling and biomass estimation\u003c/h2\u003e\n \u003cp\u003eSamples were taken daily after 24 hours for 30 days, where 20ml was filtered through Glass Fibre Carbon filters from each treatment bottle. Chlorophyl-a concetration was determined using the standard method as given in the American Public Health Association (APHA) (\u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). Extraction of the chlorophyll-a was done using acetone, where the filters and seston were folded and covered by aluminium foil and stored in a freezer overnight to aid in the bursting of the cells. The seston and the filters were then homogenised in a tissue grinder (Heidolph, 637 69, Germany) at around 5,000-rpm where 5 ml of 90% aqueous acetone was added. The samples were then transferred into a centrifuge tube, the grinder rinsed with 90% acetone (volume used was noted), and the rinsed slurry was added to the extraction slurry. The volume was then adjusted to 10 ml with 90% acetone and left for at least 8 hrs in the dark at 4\u0026deg;C for full chlorophyll-a extraction. After incubation, the samples were centrifuged for 10 min at 3,000-rpm. The clarified extract was decanted into a clean test tube. Light absorbance of the chlorophyll-a extract was measured with a spectrophotometer (Pharmacia Biotech Novaspec II, Sweden) at 750 nm and 663 nm.\u003c/p\u003e\n \u003cp\u003eChlorophyll-a concentration was calculated using, the following equation according to Talling \u0026amp; Driver (\u003cspan class=\"CitationRef\"\u003e1963\u003c/span\u003e):\u003c/p\u003e\n \u003cp\u003eBiomass; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(N=\\frac{11.4*\\left(B-A\\right)*Ve}{Vf*Lp}\\)\u003c/span\u003e\u003c/span\u003e 1\u003c/p\u003e\n \u003cp\u003eWhere; B\u0026thinsp;=\u0026thinsp;absorbance at 663 nm\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eA\u0026thinsp;=\u0026thinsp;absorbance at 750 nm\u003c/p\u003e\n \u003cp\u003eVe\u0026thinsp;=\u0026thinsp;volume of extract used (ml)\u003c/p\u003e\n \u003cp\u003eVf\u0026thinsp;=\u0026thinsp;volume of sample filtered (ml)\u003c/p\u003e\n \u003cp\u003eLp\u0026thinsp;=\u0026thinsp;light path length of the cuvette (cm)\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eAfter which the Chlorophyll-a concentration was used to derive the growth rate, divisions per day, and the generation time as critical parameters of importance. These were all derived from the equations developed by Levasseur et al. (\u003cspan class=\"CitationRef\"\u003e1993\u003c/span\u003e):\u003c/p\u003e\n \u003cp\u003eGrowth rate; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({K}^{\\text{\u0026apos;}}=\\frac{Ln\\left(\\frac{N2}{N1}\\right)}{t2-t1}\\)\u003c/span\u003e\u003c/span\u003e 2\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eWhere N1 and N2\u0026thinsp;=\u0026thinsp;biomass at time1 (t1) and time2 (t2) respectively;\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eDivisions per day and the doubling time were calculated based on the specific growth rate.\u003c/p\u003e\n \u003cp\u003eDivisions per day; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{Div}{day}=\\frac{{K}^{\\text{\u0026apos;}}}{Ln2}\\)\u003c/span\u003e\u003c/span\u003e 3\u003c/p\u003e\n \u003cp\u003eWhere Ln2\u0026thinsp;=\u0026thinsp;Natural logarithm to the base 2\u003c/p\u003e\n \u003cp\u003eDoubling time; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(Doub\\text{\u0026apos;} t =\\frac{1}{\\frac{Div}{day}}\\)\u003c/span\u003e\u003c/span\u003e 4\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section3\" id=\"Sec6\"\u003e\n \u003ch2\u003e1.3.4 Statistical analyses\u003c/h2\u003e\n \u003cp\u003eThe biomass, growth rate, divisions per day, and doubling time of \u003cem\u003eOocystis\u003c/em\u003e sp. cultured in the different treatments were compared using Sigma Plot software (version 14) through Kruskal-Wallis H test, as the data failed Normality Test (Shapiro-Wilk: P\u0026thinsp;\u0026lt;\u0026thinsp;0.050).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003cdiv class=\"Section3\" id=\"Sec8\"\u003e\n \u003ch2\u003e1.4.1 Water quality\u003c/h2\u003e\n \u003cp\u003eMean temperature during the study ranged from 24.23\u003csup\u003e0\u003c/sup\u003eC to 27.33\u003csup\u003e0\u003c/sup\u003eC. Temperature variations during the study period were not significant among the treatments (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The pH for Treatment 1 ranged from 6.8 to 7.7, where there was a steady increase from the beginning of the study. On the other hand, pH for Treatment 2 ranged from 6.6 to 7.4, and Treatment 3 ranged from 6.4 to 7.2, all following a similar trend as that of Treatment 1 characterised by an increase in the pH. Finally, the control treatment had a pH ranging from 6.1 to 7.1, also having a similar trend in time with the other three treatments\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec9\"\u003e\n \u003ch2\u003e1.4.2 Comparison of chlorophyll-a concentration at different fertilizer treatments.\u003c/h2\u003e\n \u003cp\u003eThe concentrations o of chlorophyl-a in Treatment 1 was highest (7.715\u0026thinsp;\u0026plusmn;\u0026thinsp;0.667 \u0026micro;g/ml) followed by the Control (6.963\u0026thinsp;\u0026plusmn;\u0026thinsp;0.788 \u0026micro;g/ml) and Treatment 2(6.862\u0026thinsp;\u0026plusmn;\u0026thinsp;0.617 \u0026micro;g/ml), with the least in Treatment 3 (6.441\u0026thinsp;\u0026plusmn;\u0026thinsp;0.555 \u0026micro;g/ml). There were no statistically significant differences in mean concentrations of chlorophyll-a among different treatments (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec10\"\u003e\n \u003ch2\u003e1.4.3 Temporal variations in chlorophyll-a concentration.\u003c/h2\u003e\n \u003cp\u003eGenerally, the concentration increased gradually in all treatments until it reached its peak on different days for all the treatments. The control treatment (Modified Bolds 3N Medium) achieved the highest chlorophyll-a concentration on the 25th day (12.977\u0026thinsp;\u0026plusmn;\u0026thinsp;0.788 \u0026micro;g/ml). Treatment 1 followed, reaching its highest chlorophyll-a concentration on the 17th day (11.4437\u0026thinsp;\u0026plusmn;\u0026thinsp;0.667 \u0026micro;g/ml). Treatment 2 achieved its highest concentration on the 21st day (11.3696\u0026thinsp;\u0026plusmn;\u0026thinsp;0.617 \u0026micro;g/ml), and finally Treatment 3 had the least concentration, which was achieved on the 16th day (10.2714\u0026thinsp;\u0026plusmn;\u0026thinsp;0.555 \u0026micro;g/ml). Overall, the chlorophyll-a concentration in all the treatments fluctuated once the optimum concentration was achieved. At the end of the experiment, the concentration of chlorophyll-a in the treatments followed a similar pattern, with the control having the highest (11.2442\u0026thinsp;\u0026plusmn;\u0026thinsp;0.788 \u0026micro;g/ml) while treatment 3 had the least (5.8197\u0026thinsp;\u0026plusmn;\u0026thinsp;0.555 \u0026micro;g/ml) (see Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e to \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n \u003cp\u003eThe chlorophyll-a concentration varied significantly in each treatment with time (Kruskal-Wallis H test: P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for all the treatments. At the beginning of the study, all the treatments had equal chlorophyll-a concentration (where 100 ml of stock \u003cem\u003eOocystis\u003c/em\u003e sp. cultured having a concentration of 10.0719 \u0026micro;g/ml was introduced into 2.49 litres of respective treatment media).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec11\"\u003e\n \u003ch2\u003e1.4.4 Growth rate, Divisions per day, and Doubling time\u003c/h2\u003e\n \u003cp\u003eThe maximum, mean, and least values of the growth rate, divisions per day, and doubling time in different treatments are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. There was no significant difference in the mean growth rate(Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), divisions per day (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and doubling time (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) for the treatments.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe maximum, mean, and least values of the growth rate, divisions per day, and doubling time of different treatment culture media.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment2\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrowth rate \u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.376\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.545\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrowth rate \u003csub\u003emean\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0885\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0837\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGrowth rate \u003csub\u003eleast\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.215\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDivisions per day \u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.543\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.867\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.676\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.786\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDivisions per day \u003csub\u003emean\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.121\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDivisions per day \u003csub\u003eleast\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.302\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.307\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDoubling time \u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1028.630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70.142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e187.374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.900\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDoubling time \u003csub\u003emean\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.722\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-11.267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.352\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.412\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDoubling time \u003csub\u003eleast\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-124.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-532.684\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-9.929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-39.418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec12\"\u003e\n \u003ch2\u003e1.4.5 Media Cost and Biomass\u003c/h2\u003e\n \u003cp\u003eThe control (Modified Bold 3N Medium) was produced at a cost of 11.28 KSh per litre, the other media were derived from urea, NPK and DAP at a cost of 0.14 KSh per litre for treatment 1, 0.18 KSh per litre for treatment 2 and 0.22 KSh per litre for treatment 3. Distilled water was generated by a distiller in the laboratory for use in generating the control media (Modified Bold 3N Medium) while harvested rain water was used for the experiment for treatment 1,2 and 3; hence, their cost were not captured in the above calculation.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003cp\u003eThe nature of the culture media is the main determining factor in the growth of microalgae, their productivity and ultimately their biomass as long as the pH, light intensity, and temperature needs have been fulfilled (da Silva Ferreira and Sant\u0026apos;Anna, 2017; Gani et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chowdury et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The light provided to the culture was by white light-emitting diode tubes (3200K, 9W). Csavina et al. (\u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e) illustrated that \u003cem\u003eOocystis\u003c/em\u003e sp. requires the optimum light intensity of 150 \u003cem\u003e\u0026micro;\u003c/em\u003emol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Latala et al. (\u003cspan class=\"CitationRef\"\u003e1991\u003c/span\u003e) found that the growth of \u003cem\u003eOocystis\u003c/em\u003e sp. was completely inhibited at high light intensities ranging between 270\u0026ndash;380 \u0026micro;E/m\u003csup\u003e2\u003c/sup\u003es.\u003c/p\u003e\n \u003cp\u003epH influences the quantity of free carbon in the culture media and the balance between carbonate and bicarbonate. In this study, the pH was adjusted to range between 6.0 to 8.0, as reported by RAo (\u003cspan class=\"CitationRef\"\u003e1963\u003c/span\u003e), to suit the growth of \u003cem\u003eOocystis\u003c/em\u003e sp. optimally. Mayo (\u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e) found similar results while culturing \u003cem\u003eChlorella\u003c/em\u003e sp. with the maximum growth rate at pH ranging from 6.31 to 6.84. Changes in the media pH were attributed to the photosynthetic and decomposition process of the cultured \u003cem\u003eOocystis\u003c/em\u003e sp. ( Tucker and D\u0026apos;Abramo 2008; Rahardini et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eDuring the study, the water temperature range was in optimal range as reported by Nalley et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e), where they found the best temperature for optimum growth of \u003cem\u003eOocystis\u003c/em\u003e sp. ranged between 26.60\u003csup\u003e0\u003c/sup\u003eC to 27.78\u003csup\u003e0\u003c/sup\u003eC. Chowdury et al. (\u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) state that temperature is critical in micro-algae culture as it affects the algal growth rate, cell size, biochemical composition, and nutrient requirements.\u003c/p\u003e\n \u003cp\u003eThe temporal chlorophyll-a concentration varied amongst the treatments, mainly due to the different constituents in the culture treatment media, with the differences being significant with time in all the treatments. Sutkowy et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) state that the composition of culture media influences the growth of micro-algae in vitro. Overall, all treatments followed a similar trend where there was a decrease in the chlorophyll-a concentration once it was at its highest biomass. The pattern achieved is similar to that obtained by Rahardini et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) while assessing the growth of \u003cem\u003eChlorella\u003c/em\u003e sp., which is also found in the Chlorophyceae class of Green algae.\u003c/p\u003e\n \u003cp\u003eAt the begging of the experiment, there was a steady increase (exponential phase) in the chlorophyll-a concentration due to sufficient nutrient concentration in all the treatments. There was no lag phase observed during the study, which can be attributed to the conditions of the inoculum (Spencer \u003cspan class=\"CitationRef\"\u003e1954\u003c/span\u003e). Talling (\u003cspan class=\"CitationRef\"\u003e1966\u003c/span\u003e) notes that an inoculum obtained from a healthy exponentially growing population is unlikely to have a lag phase when transferred to a freshly prepared media under similar growth conditions (light, temperature, and pH); consequently, there is no need for physiological adaptations for growth. The lack of a lag phase is crucial as it reduces the time required for upscaling the culture, allowing the harvesting of cultured cells sooner. The exponential phase is characterised by the production of materials that are also capable of growth (Fogg and Thake \u003cspan class=\"CitationRef\"\u003e1987\u003c/span\u003e), with its length depending on the size of the inoculum, the growth rate, and the capacity of the medium and culturing conditions to support algal growth.\u003c/p\u003e\n \u003cp\u003eThe exponential phase was followed by the stationary phase, characterised by a decline in population growth, resulting from reduced nutrient concentration in the culture media, causing the number of cells to remain constant. The stationary phase is characterised by zero net growth, as the rate of the growth of the cells is equal to the rate of cell death ( Nystr\u0026ouml;m \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Maier and Pepper \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Finally, the death phase follows, as nutrients in the culture media run out, resulting in the death of cultured algal cells; therefore, a decline in the chlorophyll-a concentration was witnessed. The death rate of cells in the media became higher than the rate of cell growth (Maier and Pepper \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe study demonstrated that the inorganic fertilizers\u0026apos; specific compositions and comparison between the culture media did not have any significant impact on the growth rate, divisions per day, and the doubling time of cultured \u003cem\u003eOocystis\u003c/em\u003e sp.. Similar results were also obtained by Rahardini et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) who also found no significant differences in the growth rate and doubling time of \u003cem\u003eChlorella\u003c/em\u003e sp. cultured in media with different compositions. The divisions per day obtained was higher than those from Csavina et al. (\u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e), which can be attributed to the continuous agitation of the algal cells during culture, consequently not allowing the clamping of cells together, resulting in deaths and ultimately decline in the growth rate and divisions per day. Research carried out by Sobczuk et al. (\u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e) showed an increase in the growth rate of microalgae with an increase with agitation; it should also be noted that excessive agitation can result in cell death.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003cp\u003eThis study found that inorganic fertilizers can be used as an option of cost-effective media in the culture of \u003cem\u003eOocystis\u003c/em\u003e sp., with the ratio of 3:1:1 while using NPK, urea, and DAP fertilizers respectively to achieve the highest biomass within the shortest period. The finding that there were no significant differences in the \u003cem\u003eOocystis\u003c/em\u003e sp. biomass using either medium is an encouraging result as it offers opportunities to explore the use of low-cost media which is equally competitive with the standard media in mass production of \u003cem\u003eOocystis\u003c/em\u003e sp. for potential use in fish feeds in Aquaculture.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by The Kenya Climate Smart Agriculture Project (KCSAP)\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mukoma, Marvin Gaye. The first draft of the manuscript was written by Mukoma, Marvin Gaye and all authors commented on subsequent versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgment\u003c/h2\u003e\n\u003cp\u003eThe Kenya Climate Smart Agriculture Project (KCSAP) and Biological Sciences Department, Egerton University.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmerican Public Health Association (APHA) (2005) Standard method for the examination of water and wastewater 21\u003csup\u003est\u003c/sup\u003e edition. Washington DC: America Water Works Association and Water Control Federation. \u003c/li\u003e\n\u003cli\u003eAnsari FA, Singh P, Guldhe A, Bux F (2017) Microalgal cultivation using aquaculture wastewater: integrated biomass generation and nutrient remediation. 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Modified Bold 3N Medium\u003cem\u003e.\u003c/em\u003e Accessed on 20/4/2021, from https://utex.org/products/modified-bold-3n-medium?variant=30991514763354 \u003c/li\u003e\n\u003cli\u003eWaithaka PN, Gathuru EM, Githaiga BM, Kimani SN (2017) Control of passion fruit fungal diseases using essential oils extracted from rosemary \u003cem\u003e(Rosmarinus officinalis\u003c/em\u003e) and eucalyptus (\u003cem\u003eEucalyptus agglomerata\u003c/em\u003e) in Egerton University Main Campus Njoro, Kenya. Int J Microbiol \u003cem\u003e2017\u003c/em\u003e,1-6. https://doi.org/10.1155/2017/2814581 \u003c/li\u003e\n\u003cli\u003eWin TT, Barone GD, Secundo F, Fu P (2018) Algal biofertilizers and plant growth stimulants for sustainable agriculture. Ind Biotechnol \u003cem\u003e14\u003c/em\u003e(4), 203-211. https://doi.org/10.1089/ind.2018.0010 \u003c/li\u003e\n\u003cli\u003eYaakob MA, Mohamed RMSR, Al-Gheethi A, Ravishankar GA, Ambati RR (2021) Influence of nitrogen and phosphorus on microalgal growth, biomass, lipid, and fatty acid production: an overview. Cells \u003cem\u003e10\u003c/em\u003e(2), 393. https://doi.org/10.3390/cells10020393 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Oocystis sp., Mass production, Inorganic fertilizers, Climate smart","lastPublishedDoi":"10.21203/rs.3.rs-1637529/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1637529/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUse of microalgae as source of food in aquaculture production is gaining recognition due to their rapid growth rate that promises high biomass generation within a short time. The challenge faced is getting good and inexpensive nutrients source to be used in mass production of the required microalgae. This study investigated the effect of different nutrient combination in influencing the growth rate of the of the green algae \u003cem\u003eOocystis\u003c/em\u003e sp. which has been indentified as possible protein source for the raising of \u003cem\u003eOrechromis niloticus\u003c/em\u003e fingerlings for fish farming. Modified Bolds 3N Medium and commercial agricultural fertilisers (urea, NPK and DAP) media were compared to establish the appropriate combination that would result into high biomass generation but at the lowest cost possible. The Modified Bold 3N Medium acted as the control, at a cost of 11.28 KSh per litre, the other media were derived from urea, NPK and DAP (varying the ratio of each) at a cost of treatment 1 (0.14 KSh per litre), treatment 2 (0.18 KSh per litre) and treatment 3 (0.22 KSh per litre). The algae was cultured for five weeks with samples taken daily for biomass analyses using chloropyhll-a concentration as the surrogate for \u003cem\u003eOocystis\u003c/em\u003e sp. biomass for 30 days, from each treatment was determined. The growth rate, doubling time, and divisions per day were then estimated based on this chlorophyll-a concentration. The results showed that the mean concentrations of chlorophyll-a in treatment 1 was highest (7.715\u0026thinsp;\u0026plusmn;\u0026thinsp;0.667 \u0026micro;g/ml) while treatment 3 (6.441\u0026thinsp;\u0026plusmn;\u0026thinsp;0.555 \u0026micro;g/ml) had the least. There was no significant differences in the mean concentrations of chlorophyll-a in the four treatments (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The chlorophyll-a concentration varied significantly in each treatment with time (Kruskal-Wallis H test: P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was no significant difference in the growth rate (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), divisions per day (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and doubling time (Kruskal-Wallis H test: P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from the different treatments. The results of this study showed that inorganic fertilizers can be used as cost-effective media in the mass scale culture of \u003cem\u003eOocystis\u003c/em\u003e sp.\u003c/p\u003e","manuscriptTitle":"Sustainable Fish Feeds: Optimization of Levels of Inorganic Fertilizers for Mass Production of Oocystis Sp. For Climate Smart Aquaculture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-16 15:11:09","doi":"10.21203/rs.3.rs-1637529/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2022-09-20T18:04:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-05T02:25:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e2baa7ea-2b3c-4613-80cb-ddb0df6b4383","date":"2022-07-23T05:37:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-18T12:00:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f7f5cae8-5a46-45aa-b7ae-d9e2bf734df5","date":"2022-06-17T17:25:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-17T14:52:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-13T06:11:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-05-10T10:17:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2022-05-09T09:05:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c5ab2444-1b13-4631-bb57-d36c63c212dd","owner":[],"postedDate":"May 16th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-02-23T18:33:42+00:00","versionOfRecord":{"articleIdentity":"rs-1637529","link":"https://doi.org/10.1007/s10499-022-00986-5","journal":{"identity":"aquaculture-international","isVorOnly":true,"title":"Aquaculture International"},"publishedOn":"2022-10-01 00:00:00","publishedOnDateReadable":"October 1st, 2022"},"versionCreatedAt":"2022-05-16 15:11:09","video":"","vorDoi":"10.1007/s10499-022-00986-5","vorDoiUrl":"https://doi.org/10.1007/s10499-022-00986-5","workflowStages":[]},"version":"v1","identity":"rs-1637529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1637529","identity":"rs-1637529","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}