Reproduction rate and microbial load of the rotifer Brachionus calyciflorus (Pallas, 1766) fed on Chlorella sp. cultured on organic media

Reproduction rate and microbial load of the rotifer Brachionus calyciflorus (Pallas, 1766) fed on Chlorella sp. cultured on organic media | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reproduction rate and microbial load of the rotifer Brachionus calyciflorus (Pallas, 1766) fed on Chlorella sp. cultured on organic media Solomon Melaku, Abebe Getahun, Seyoum Mengestou, Akewake Geremew, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4690614/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract An experiment was conducted to evaluate the culture performance and total microbial load of the rotifer Brachionus calyciflorus reared on microalgae Chlorella sp . cultured with a replacement of 50% of the standard Bolds’ Basal Medium (BBM) with poultry manure filtrate (POMF), sheep manure filtrate (SHMF), water hyacinth root compost filtrate (WHRCF), water hyacinth leaf compost filtrate (WHLCF), water hyacinth root and leaf compost filtrate (WHR + LCF) and a control treatment with 100% BBM. The results indicated that the POMF substituted culture of Chlorella sp . gave significantly highest cell density (1.83x10 8 cells mL − 1 ) followed by the 100% BBM culture (1.74x10 8 cells mL − 1 ) supporting 272 ± 6.4 and 122.2 ± 5.53 rotifers mL − 1 B. calyciflorus population, respectively. The SHMF culture gave the third highest Chlorella sp. density (5.94x10 7 cells mL − 1 ) and supports 83.4 ± 13.78 rotifers mL − 1 B. calyciflorus population. The three treatments with the compost filtrate of the different parts of water hyacinth gave the least Chlorella sp . cell density and B.calyciflorus population as well. In terms of the total viable bacterial count of B.calyciflorus cultured on the substitution of the different organic media indicated that B.calyciflorus cultured on 100% BBM and 50% POMF- based culture gave significantly lowest total bacterial counts of 6.9x10 6 ± 9.9x10 5 and 8.05x10 6 ± 4.94x10 5 CFU mL − 1 , respectively while SHMF, WHR + LCF, WHLCF, WHRCF group showed higher total bacterial counts of 1.62x10 7 ± 2.83x10 5 , 1.09x10 7 ± 7.78x10 5 , 1.25x10 7 ± 7.07x10 4 , 1.03x10 7 ± 7.07x10 4 CFU mL − 1 , respectively in non-rinsed samples. Therefore, it can be concluded that 50% substitution the standard BBM media for Chlorella sp. culture with organic manures such as POMF can be a sustainable alternative to culture the rotifer B. calyciflorus for larval fish culture. Live feed Microbial load Organic media Rotifer culture Figures Figure 1 Figure 2 1. Introduction Brachionus calyciflorus is one of the live feed organisms used in the larviculture of freshwater finfishes (Lim & Wong 1997 ; Aoyama et al. 2015 ; Ekelemu 2015 ). This rotifer has been a good substitute for the costly Artemia cyst import as it is locally available for most resource limited developing countries (Arimoro 2006 ; Ekelemu 2015 ; Melaku et al. 2022 ). In addition, due to its relative small size, B.calyciflorus is used to wean ornamental fish larvae with small mouth gape during their initial feeding, as they are unable to ingest the larger-sized Artemia nauplii (Lim & Wong 1997 ; Lim et al. 2003 ; Lahnsteiner et al. 2023 ). B.calyciflorus is also used successfully in the weaning of African catfish larvae in different settings (Awaïss & Kestemont 1998 ; Evangelista et al. 2005 ; Abaho et al. 2016 ; Kwikiriza et al. 2016 ). However, the mass culture of B.calyciflorus is hindered by a lack of suitable artificial diets to substitute the costly microalgae, which adds extra cost to the larviculture of commercially important fish species. Microalgae have been an indispensible part of rotifer culture systems providing stable and clean cultures (Lavens and Sorgeloos 1996 ; Nagappan et al. 2021 ). Moreover, the nutritional composition of rotifers cultured on microalgae has superior quality compared to those fed with artificial diets (Dhert et al. 2001 ). microalgae are primarily utilized in live feed culture to transfer essential nutrients, such as polyunsaturated fatty acids (PUFA) to the finfish larvae (Conceição et al. 2010 ; Richmond and Hu 2013 ; Watanabe et al. 2016 ). Despite their benefits, the widespread application of microalgae in aquaculture is hindered by the costly synthetic media usage and complex culture systems required (Chauton et al. 2015 ). Therefore, alternatives to the costly synthetic media in the form of either total or partial substitution with different forms and pre-processing of organic manure resources have been an area of research recently (Asmare et al. 2014 ; Tan et al. 2018 ; Zhou et al. 2019 ; Bauer et al. 2021 ). These organic manure resources are readily available and have the potential to replace totally or partially the costly synthetic media if properly processed and applied (Bjornsson et al. 2013 ; Wang et al. 2020 ). On the other hand, application of these manure resources for microalgae culture has an implication of reducing the impacts of the resources on the environment if disposed directly. Due to this fact most applications of this manure filtrates in microalgae culture are mainly aimed at reclaimng the organic wastes to mitigate their environmental impacts and enhancing lipid production of some microalgae species for biodiesel production (Wang et al. 2010 ; Pszczółkowska et al. 2019 ). Chlorella sp. , especially when grown mixotrophically and heterotrophically, demonstrate robust growth rates and high biomass productivity, which is essential for industrial applications.These species are capable of accumulating significant amounts of intracellular lipids, making them suitable for biofuel production. The lipid content can range from 20–70% of cell weight, leading to high yields. The potential for nutrient recycling in heterotrophic microalgae cultures, which can reduce the demand for virgin nutrients and water, thus enhancing the sustainability of the production process. Chlorella sp. have been successfully used in various commercial applications, including nutritional supplements and high-value fatty acids, due to their desirable qualities (Lowrey et al. 2016 ). While organic manure filtrates have been primarily used to reclaim organic wastes and enhance lipid production, limited research has explored their application in microalgae culture for live feed production (Dahril 1998 ; Ogello et al. 2019 ; Zhu et al. 2024 ). This is partly due to biosafety concerns associated with the potential presence of pathogenic microorganisms in organic manure, which could pose risks to aquaculture species fed on live feed. High organic matter load in rotifer culture tanks, in combination with high temperature, favors bacteria to proliferate (Skjermo and Vadstein 1993 ), some of which may be pathogenic to rotifers(Yu et al. 1990 ). Although most bacteria have no detrimental effect on rotifers themselves, the real concern lies in the risk to host (larvae) feeding on the rotifers (Skjermo and Vadstein 1993 ; Yan et al. 2007 ). However, the organic manure resources could be an alternative source of nutrients for microalgae culture for live feed production and green water aquaculture systems in general with proper pre-processing and culture system combinations in resource-limited developing countries such as Ethiopia where organic manure resources are abundant (Samani 2007 ). Therefore, this study aims to evaluate the effect of substituting costly synthetic media with different organic media resources on microalgae culture performance and its subsequent effect on the culture performance of B. calyciflorus as live feed for larval fish culture. 2. Materials and methods 2.1 Experimental set up The experiment was conducted at the Phycology laboratory of the Department of Zoological Sciences of Addis Ababa University. The experiment acknowledges the inherent differences in nutrient composition of the organic media derived from different sources. This variability is expected to impact the growth performance of the microalgae and rotifers. The study attempts to standardize conditions by autoclaving the organic media to eliminate pathogens and storing them under controlled temperatures. In addition, carbon to nitrogen ratio was maintained at 24 for all treatments according to the recommendations by Gao et al. ( 2019 ) through serial dilution with distilled water before being used as a substitute to the BBM. The experiment compares the growth performance of microalgae and rotifers across different treatments, providing insights into the suitability of each organic media as a BBM substitute. In the first phase of the experiment, Chlorella sp. culture was conducted with a replacement of 50% of Bold’s Basal Media (BBM) with five organic media and a control with 100% standard BBM in duplicate flasks for 10 days. The experimental treatments were poultry manure filtrate (POMF), sheep manure filtrate (SHMF), water hyacinth root compost filtrate (WHRCF), water hyacinth leaf compost filtrate (WHLCF), water hyacinth root and leaf compost filtrate (WHR + LCF) and a control treatment with 100% BBM. At the eleventh day of the culture period, B.calyciflorus rotifer was inoculated at a density of 20 rotifers mL -1 to each culture flask. The rotifer culture was kept for 4 days to evaluate the capacity of the Chlorella sp. cultured in the different culture media on the rotifer reproduction rate. At the end of the rotifer culture period, rotifers were enumerated and compared among the different treatments for their carrying capacity. 2.2 Microalgae strain and culture conditions Chlorella sp. with accession number EMCC-M31 Lake Beseka strain was provided by the Ethiopian Biodiversity Institute, Addis Ababa, Ethiopia. The Chlorella sp. stock was inoculated in a 250 mL flask with 200 mL of BBM media at pH 8. The microalga was subsequently up scaled to cell densities appropriate to start the experiment. Then 50% of the Bolds’ Basal Medium (BBM) was substituted with the respective organic media except for the control with 100% BBM culture. The culture was run for 10 days using 1L Erlenmeyer flasks with 800mL culture volume. The microalgae culture was run mixotrophically under a culture chamber with 27 µmol m -2 s -1 light intensity. The photo period was set at 16 to 8 light and dark periods respectively. The daily cell growth rate was monitored using optical density measurement at 750nm and 686nm (Chioccioli et al. 2014 ) (Fig. 1 a,b). The Chlorella sp. growth rate was calculated using the following equation: $$\:\:\:\:\:\:\:\:\:{\mu\:}\:=\:\:\text{l}\text{n}(n1/n2)/(t2-t1)$$ Where µ is the specific growth rate (day − 1 ), n1 and n2 are initial and final cell density counts (cells/mL), while t1 and t2 are initial and final culture periods in days. The micro algal cell counts were carried out at the start and last dates of the culture using Sedgwick rafter cells. Bold’s Basal Medium (BBM), was prepared from 10mL per liter stock solutions of 25 g L -1 NaNO 3 , 2.5 g L -1 of CaCl 2 .2H 2 O, 7.5 g L -1 of MgSO 4 .7H 2 O, 7.5 g L -1 of K 2 HPO 4 , 17.5 g L -1 of KH 2 PO 4 , 2.5 g L -1 of NaCl and 1 mL per liter of 50 g L -1 EDTA anhydrous, 31 g L -1 of KOH, 4.98 g L -1 of FeSO 4 .7H 2 O, 1 mL of H 2 SO 4 , 11.4 g L -1 of H 3 BO 3 , 8.82 g L -1 ZnSO 4 .7H 2 O, 1.44 g L -1 of MnCl 2 .4H 2 O, 0.71 g L -1 of MoO 3 , 1.57 g L -1 of CuSO 4 .5H 2 O, 0.49 g L -1 of Co(NO 3 ) 2 .6H 2 O. The initial pH of the medium was adjusted to 8. 2.3 Organic media processing and preparation Poultry and sheep manures used in the experiment were obtained from Asko and Winget area small scale poultry and sheep markets in Addis Ababa while the water hyacinth compost filtrate were obtained from an experimental field at Batu fisheries and other aquatic life research center, Oromia Agricultural Research Institute. The samples were collected in August 2022. Organic media were dried immediately to a constant weight over sunlight and ground to a fine particulate matter using a laboratory grinder. Afterwards, 1 kg of each organic manure was soaked with 3 liter distilled water overnight. Then, the filtrates were decanted and filtered with 30µm sieve to a constant volume of 1 liter. Afterwards the organic media were autoclaved at 121 0 c for 15 minutes to remove potential pathogenic microorganisms and zooplankton. The organic media were stored in a refrigerator below − 4 0 c until being applied to the experiments and chemical composition analysis. The chemical composition of the organic media was measured photo metrically using (APHA 1995 ) standard methods (Table 1 ). Table 1 Summary of the physico-chemical properties of the constituents of the culture media POMF SHMF WHRCF WHLCF WHR + LCF TP (mg/L) 2.87 1.29 0.83 0.18 0.47 NH 3 -N (mg/L) 1.60 1.37 1.32 0.63 0.76 NO 2 -N (mg/L) 0.10 0.13 0.20 0.13 0.16 NO 3 -N (mg/L) 1.87 2.01 1.67 2.68 2.31 C:N ratio 18 33 33 26 34 TK (mg/L) 25.23 18 12.56 10.83 14.13 TSS (mg/L) 253 788 130.67 201.45 167.6 TDS (mg/L) 939 1992 1960 1012 1275 Cond (µS/cm) 2250 6010 4950 1764 1804 Turb (NTU) 244 724 312 359 159 PH 8.35 8.55 7.65 8.3 8.15 Where, POMF = poultry manure filtrate, SHMF = sheep manure filtrate, WHRCF = water hyacinth root compost filtrate, WHLCF = water hyacinth leaf compost filtrate, WHR + LCF = water hyacinth root and leaf compost filtrate The samples were analyzed following the standard methods described in (APHA 1995 ). To briefly describe the procedures, TP contents of the culture media was measured by the ascorbic acid technique after digestion with persulfate. Nitrate (NO 3 -N) was measured with the sodium salicylate method, while ammonia (NH 3 + NH 4 + - N) was determined by the phenate method. Nitrite (NO 2 -N) was determined by diazotization with Sulphanilamide and coupling to Naphthylethylene diamine di-HCl. Total Nitrogen was determined by the persulfate digestion method while total Potassium concentration was measured using Tetraphenylborate method using DR6000 HACH Spectrophotometer. Total suspended solid (TSS) was determined gravimetrically after titration of a known volume of organic media sample. The COD of the samples was determined by digesting the samples in an aluminum heating block for about 7 minutes. The Carbon to Nitrogen ratio was calculated by dividing the COD values by the total nitrogen values of the respective samples. 2.4 Rotifer strain and culture conditions “Brachionus calyciflorus Pallas 1766” were isolated from Lake Tinishu Abaya, Gurage zone, Ethiopia (plate 1a). Lake Tinishu Abaya is situated in the rift valley system of Ethiopia at 7 0 29'03.65" N latitude and 38 0 03'17.79" E longitude (Fig. 1 ). The lake covers a surface area of 1253 hectares with maximum and mean depths of 1.5 and 1.1 m respectively (Yirga and Brook 2018 ). To briefly describe the procedures, mixed population of zooplankton samples were collected by plankton net of 15 µm towed horizontally over the lakes surface several times. Several liters of lake water was filtered with the plankton net to obtain significant amount of zooplankton including B. calyciflorus and concentrated to a four liter sample collection bottles. Subsequent sieving of the samples was done over 300 and 600 µm size sieves to remove bigger zooplanktons and particulate matter. Afterwards, the samples were transported to Phycology Laboratory of the Department of Zoological Sciences, Addis Ababa University. Water physico-chemical parameters were recorded onsite with portable multi parameter probe (HACH hd401dn, Loveland, USA) (Data not presented). Up on arrival to the laboratory, confirmation of the presence of B.calyciflorus was checked under compound microscope at magnifications of 10x and 20x. One ml of sample was treated with a drop of lugol’s solution for better resolution and identification of the B.calyciflorus was done according to Koste (1978) (Plate 1a). Serial dilution method was used to isolate the B.calyciflorus from other rotifers of nearly similar size as the population density of the B.calyciflorus was dominant. The rotifer stock culture was initially conducted in filtered water brought from the lake and gradually changed to reconstituted hard water composed of the following chemicals: 96 mg NaHCO 3 , 60 mg CaSO 4 .2H 2 O, 60 mg MgSO 4 , and 4 mg KCL in one liter of distilled water according to Lavens & Sorgeloos ( 1996 ). The rotifers were cultured on Chlorella sp. at a density of 1x10 7 cells mL − 1 and a temperature of 25 0 C with a photo period of 12hr light and 12hr dark (Rico-martinez and Dodsonb 1992 ). Afterwards the rotifer cultures were up scaled subsequently until sufficient numbers were obtained to start the experiment. At the end of the four days culture, rotifers were harvested using 75 µm sieve and rotifer reproduction rate was calculated using the following formula: $$\:\:\:\:\:\:\:\:\:{\mu\:}\:=\:\:\text{l}\text{n}(n1/n2)/(t2-t1)$$ Where µ is the specific growth rate (day − 1 ), n1 and n2 are initial and final cell density counts (cells mL − 1 ), while t1 and t2 are initial and final culture periods in days 2.4 Total bacterial counts Two types of samples were taken for the total bacterial counts i.e. samples taken before the rotifers were rinsed with autoclaved tap water and samples taken after rinsing the rotifers with autoclaved tap water. One milliliter sample of rotifers along with culture water was taken from each replicate Erlenmeyer flasks in both cases. The bacterial culture was conducted at Mycology laboratory of the Department of Zoological Sciences, Addis Ababa University. Serial dilutions of the samples were made by transferring 0.1 mL of experimental samples to 0.9 mL of sterile freshwater and a pretest was conducted to determine the countable ranges before the whole samples were seeded on the petri plates. Afterwards, the appropriate dilutions were plated on Tryptic Soy Agar (TSA, HiMedia, India) in duplicates and incubated for 48 hrs at 37 0 c. After 48 hrs incubation, plates with distinct colonies in the range of 30 to 400 colony forming units were counted and the results were expressed as CFU mL − 1 (plate 2). All these procedures were carried out under sterile conditions to prevent contamination and to obtain accurate results. 2.4 Water quality parameters Culture water temperature, dissolved oxygen, pH, total ammonia nitrogen, nitrite and nitrate were measured daily during the four day rotifer culture. Portable pH and DO meters were used to measure the respective parameters in-situ . Colorimetric method was applied to measure the concentration of TAN, NO 2 + -N and NO 3 + -N in mg L − 1 using API freshwater master test kit (API®, Pennsylvania, USA). 2.5 Data analysis All the experimental data were analyzed using OriginPro learning edition release 2024 (Originlab, USA). Data were evaluated for homogeneity of variances using Levene's test. When the data were found to follow normal distribution and to be homoscedastic, final day rotifer densities, micro algal cell densities, micro algal growth rate and total bacterial counts of rinsed and non-rinsed rotifers were compared between treatments using one-way analysis of variance (ANOVA), followed by Tukey HSD test for multiple comparisons of means. However, when these assumptions were not fulfilled, Kruskal - Wallis rank Sum test was applied to compare means. 3. Results 3.1 Effects of partial substitution of BBM with organic media on the growth of Chlorella sp. The results of this experiment indicated that 50% substitution of BBM by POMF gave the highest final day cell density followed by the control with 100% BBM, but there was no significant difference ( p < 0.05) in algal growth rate between the two groups (Table 2 ). SHMF substituted cultures gave the third highest Chlorella sp. growth in terms of cell density at the last date of the culture period. On the other hand, the three treatment groups with water hyacinth-based compost filtrate gave significantly the lowest Chlorella sp. cell density and growth rate compared to the POMF and BBM culture groups. There was no significant difference in cell density between the three water hyacinth-based compost filtrates that substituted 50% BBM. Table 2 Chlorella sp. growth on organic media substituted cultures Initial cell density (cells/mL) Final cell density (cells/mL) Growth rate (Day − 1 ) p-value BBM 2.63x10 6 ± 1.55x10 4 1.74x10 8 ± 1.44x10 6b 0.42 ± 0.0014 a P < 0.0001 POMF 2.63x10 6 ± 2.69x10 4 1.83x10 8 ± 4.03x10 6a 0.42 ± 0.0032 a SHMF 2.63x10 6 ± 1.13x10 4 5.94x10 7 ± 1.65x10 6c 0.31 ± 0.0032 b WHRCF 2.66x10 6 ± 5.23x10 4 1.49x10 7 ± 1.14x10 5d 0.17 ± 0.0012 c WHLCF 2.63x10 6 ± 1.98x10 4 1.25x10 7 ± 5.09x10 4d 0.15 ± 0.0012 d WHR + LCF 2.64x10 6 ± 3.25x10 4 8.37x10 7 ± 3.82x10 4d 0.12 ± 0.0007 e Values are mean ± SD, n = 2, values with different superscript letters in the same column are significantly different at p < 0.05, Tucky HD test. Where, BBM = 100% Bolds Basal Medium, POMF = poultry manure filtrate, SHMF = sheep manure filtrate, WHRCF = water hyacinth root compost filtrate, WHLCF = water hyacinth leaf compost filtrate, WHR + LCF = water hyacinth root and leaf compost filtrate. 3.2 Effects of Chlorella sp. cultured on different organic media on the population growth of B.calyciflorus POMF substituted cultures of the microalgae gave significantly higher rotifer reproduction at the last date of the rotifer batch culture (272 ± 6.4 individuals mL − 1 ) followed by the 100% BBM (122.2 ± 5.53 individuals mL − 1 ) culture (Table 3 ). On the other hand, SHMF (83.4 ± 13.78 individuals mL − 1 ) gave the third highest rotifer reproduction (p < 0.05). The three treatments with the different water hyacinth-based composts gave the lowest rotifer reproduction corresponding with the lowest Chlorella sp . growth performance mentioned in section 3.1 . Table 3 Effects of Chlorella sp. cultured on different organic media on the population growth of B.calyciflorus Initial rotifer density (individuals mL − 1 ) Final rotifer density (individuals mL − 1 ) Rotifer growth rate (Day − 1 ) BBM 19.5 ± 1.94 122.2 ± 5.53 b 0.46 ± 0.014 b POMF 19.4 ± 1.86 272 ± 6.4 a 0.67 ± 0.029 a SHMF 20.4 ± 1.37 83.4 ± 13.78 c 0.35 ± 0.025 c WHRCF 20.1 ± 0.14 49.3 ± 1.88 d 0.22 ± 0.008 d WHLCF 20 ± 1.77 34.8 ± 1.41 e 0.14 ± 0.01 e WHR + LCF 19.9 ± 0.75 28.7 ± 2 e 0.10 ± 0.03 e p-value p < 0.0001 p < 0.0001 Values are mean ± SD, n = 2, values with different superscript letters in the same column are significantly different at p < 0.05, Tucky HSD test. Where, BBM = 100% Bolds Basal Medium, POMF = poultry manure filtrate, SHMF = sheep manure filtrate, WHRCF = water hyacinth root compost filtrate, WHLCF = water hyacinth leaf compost filtrate, WHR + LCF = water hyacinth root and leaf compost filtrate 3.3 Microbial loads of B.calyciflorus fed on Chlorella sp. cultured with organic media substitution The total bacterial counts of the non-rinsed rotifers significantly differed between treatments. The total viable bacterial load of non-rinsed rotifer cultures with POMF (8.05x10 6 ± 4.94x10 5 CFU mL -1 ) and 100% BBM (6.9x10 6 ± 9.9x10 5 CFU mL -1 ) was significantly lower compared to the other groups while the SHMF (1.62x10 7 ± 2.83x10 5 CFU mL -1 ) substituted culture was significantly higher (p < 0.05). The water hyacinth compost filtrate substituted groups gave also higher total viable bacterial counts compared to the POMF and 100% BBM culture but lower as compared to the SHMF substituted groups (Table 4 , plate 2). The total viable bacterial count of rinsed rotifers also showed significant difference amongst the different treatments and the control as well. To this end, the 100% BBM gave significantly lowest total bacterial count (7.95x10 3 ± 9.19x10 2 CFU mL -1 ) of rinsed rotifers compared to the organic media substituted groups. On the other hand, the WHR + LCF gave significantly higher total bacterial count of rinsed rotifers compared to the other groups (p < 0.05). Table 4. Microbial loads of B.calyciflorus fed on Chlorella sp. cultured with substitution of BBM with organic media. Total bacterial count (Non-rinsed) (CFU mL − 1 ) Total bacterial load (Rinsed) (CFU mL − 1 ) P-value BBM 6.9x10 6 ± 9.9x10 5d 7.95x10 3 ± 9.19x10 2c P < 0.0001 POMF 8.05x10 6 ± 4.94x10 5cd 2.44x10 4 ± 1.63x10 3b SHMF 1.62x10 7 ± 2.83x10 5a 2.82x10 4 ± 2.19x10 3ab WHRCF 1.09x10 7 ± 7.78x10 5b 2.63x10 4 ± 9.89x10 2ab WHLCF 1.25x10 7 ± 7.07x10 4b 2.92x10 4 ± 1.13x10 3ab WHR + LCF 1.03x10 7 ± 7.07x10 4bc 3.05x10 4 ± 8.48x10 2a Values are mean ± SD, n = 2, values with different superscript letters in the same column are significantly different at p < 0.05, Tucky HSD test. Where, BBM = 100% Bolds Basal Medium, POMF = poultry manure filtrate, SHMF = sheep manure filtrate, WHRCF = water hyacinth root compost filtrate, WHLCF = water hyacinth leaf compost filtrate, WHR + LCF = water hyacinth root and leaf compost filtrate 3.4 Water quality parameters Basic water quality parameters such as temperature, pH, DO and nitrogenous compounds (TAN, NO 2 -N and NO 3 -N) were measured daily during the four day rotifer culture whereas the measured values at the last date of the culture period are presented in Table 5 below. Table 5 Water quality parameters measured at the final date of rotifer culture WHRCF WHLCF WHR + LCF POMF SHMF BBM Temperature (0 c ) 24.5 ± 1.1 25 ± 1.2 25.1 ± 1.2 24.8 ± 1.6 25.2 ± 1.1 24.9 ± 1.3 pH 8.05 ± 0.21 8.2 ± 0.14 8.15 ± 0.07 8.35 ± 0.07 8.05 ± 0.07 8.55 ± 0.07 DO (mg L -1 ) 3.75 ± 0.35 4.05 ± 0.21 3.85 ± 0.35 4.65 ± 0.63 3.35 ± 0.21 4.2 ± 0.14 TAN (mg L -1 ) 0.18 ± 0.08 0.19 ± 0.08 0.18 ± 0.08 0.37 ± 0.17 0.19 ± 0.08 0.18 ± 0.08 NO 2 + -N (mg L -1 ) 0.18 ± 0.08 0.31 ± 0.08 0.18 ± 0.09 0.19 ± 0.08 0.37 ± 0.17 0.18 ± 0.08 NO 3 + -N (mg L -1 ) 2.25 ± 0.35 2.5 ± 0.71 2.75 ± 0.35 2.5 ± 0.71 3.25 ± 1.06 2.25 ± 0.35 Values are mean ± SD, n = 2, Where, BBM = 100% Bolds Basal Medium, POMF = poultry manure filtrate, SHMF = sheep manure filtrate, WHRCF = water hyacinth root compost filtrate, WHLCF = water hyacinth leaf compost filtrate, WHR + LCF = water hyacinth root and leaf compost filtrate 4. Discussion 4.1 Chlorella sp . culture performance on different organic media substitutions The culture performance in terms of culture density, growth rate and optical density measurement at the final day of the culture period indicated that POMF-substituted cultures achieved significantly higher cell density compared to other treatments, followed by the 100% BBM. This result is in agreement with the findings of Markou et al. ( 2016 ) where Chlorella vulgaris showed significantly higher biomass production on raw poultry litter leachate-based media compared to the standard BG-11 media. This might be partly due to the ability of the Chlorella sp . to grow mixotrophicaly which is an attribute of the species to grow in light limited, murky media which most microalgae cannot tolerate (Heredia-Arroyo et al., 2011 ; Bansal, 2019 ). One critical challenge in using organic manure as a culture medium for microalgae is the high amount of suspended solids that blocks light penetration, a critical component in algae culture setups (Wang et al. 2020 ). In addition, the notable effect of supporting higher growth of Chlorella sp. can be attributed to the relatively higher levels of major nutrients such as Phosphorus and Potassium in the poultry manure filtrate compared to other treatments (Table 1 ). Agwa & Abu ( 2014 ) found that Chlorella sp. cultured in poultry manure medium showed higher lipid production, achieving nearly 18% (w/w) lipid content under sunlight as light source. Rajagopal et al. ( 2021 ) reported that Chlorella vulgaris was able to grow and utilize nutrients from a 10% diluted chicken manure digestate with better cell growth rate and biomass productivity. Another study by Dincă et al. ( 2021 ) indicated that Chlorella spp . produced higher biomass compared to the BG-11 standard media. On the other hand, the sheep manure filtrate gave the third highest microalgal cell density despite its nutrient composition competing with the poultry manure filtrate and 100% BBM control. This might be attributed to the heavily dark coloration of the SHMF which inhibited complete light blockage which shifts the mode of microalgae culture from mixotrophic which Chlorella sp. performed better to heterotrophic culture mode (Liang et al. 2009 ; Mohammad Mirzaie et al. 2016 ). In addition, there were bubble formations with the SHMF culture setups during aeration which might affect the growth of the microalgae (plate 1c left end). Three treatments with the different parts of water hyacinth compost filtrate gave the least Chlorella sp. growth as indicated in Table 2 . This is partly due to the suboptimal nutrient composition compared to the other treatments and the heavy dark coloration of the organic media even worse than the SHMF as mentioned above (plate 1c right). Although Water hyacinth compost application is more commonly reported in soil amendment in crop production (Malik 2007 ; Sagar et al. 2019 ), few studies report its application in microalgae culture (Iba et al., 2019 ; Dahiya et al., 2021 ). Iba et al. ( 2019 ) reported that Chlorella vulgaris cultured at 5% water hyacinth liquid fertilizer gave better cell density, specific growth rate and biomass production in 40 mg L -1 salinity level. Conversely, Dahiya et al. ( 2021 ) found that water hyacinth hydrolysate, combined with inorganic carbon and a 12:12 hour light-dark photoperiod, produced the highest biomass of nearly 200 mg/L in two strains of Chlorella sorokiniana isolated from municipal wastewater. 4.2 B.calyciflorus culture performance on the Chlorella sp. cultured under substitution of algal growth media with different organic media The B.calyciflorus culture performance in terms of rotifer growth rate and population density at the last date of the culture period followed the same trend of the algal culture performance. The Chlorella sp. Culture with POMF substituted group gave significantly higher B. calyciflorus growth rate and population density followed by the 100% BBM culture. This result is in agreement to the reports of Dahril ( 1998 ) in which chicken manure gave the highest cell density of Chlorella spp. and subsequently supported higher B.calyciflorus population compared to duck, quail, buffalo and horse manures. The author suggested that high nitrogen content of the chicken manure medium contributed to this effect. In another study by Ogello & Hagiwara, ( 2015 ) chicken manure extract at 2 mL L -1 supplied simultaneously with Chlorella vulgaris in the culture jars gave significantly higher B.calyciflorus population growth rate and population density. A more recent study by Zhu et al. ( 2024 ) indicated that 10mL L -1 of chicken manure extract cultured Chlorella vulgaris promoted the growth rate and changed the life history of the rotifer Brachionus plicatilis compared to the standard BG-11 medium. 4.3 Bacterial load of B.calyciflorus reared on Chlorella sp. cultured on different organic media The main challenges in the application of organic manure resources in live feed culture setups is the potential risk of introducing pathogenic microorganisms into the culture system and to the fish larvae feeding on the live feeds. Rotifer culture systems are characterized with high organic matter load and when this organic matter load is accompanied by high culture temperature, the resultant effect creates high bacterial load in the culture water and rotifers. Since rotifers are filter feeders which can ingest bacteria, the bacterial community of rotifers is similar to the bacterial community of the culture water (Starkweather et al. 1979 ; Seaman et al. 1986 ; Skjermo and Vadstein 1993 ). Rotifers were found to be the major vectors of pathogenic bacteria for fish larvae in previous studies (Verdonck et al. 1997 ; Yan et al. 2007 ). Therefore, decontamination of organic manure resources through different techniques is crucial from the biosecurity point of view. To this end, the organic manure resources applied in this study were autoclaved at 121 0 c for 15 minutes before being applied to the Chlorella sp. culture and its application for B.calyciflorus culture. Afterwards, the bacterial load of B.calyciflorus was evaluated using the classical culture based method in rinsed and non-rinsed rotifers with an autoclaved tap water. Use of sodium nifurstyrenate with bacterial reduction rate of 10 to 100 times have been reported by Tanasomwang & Muroga ( 1992 ) where the microbial community composition shifted to less Vibrio , but higher Pseudomonas. Munro et al. ( 1999 ) found that UV light could reduce the bacterial load by more than 90% and Suantika et al. ( 2001 ) used ozone in a rotifer recirculation system to reduce bacterial densities. However, microbial composition changes were not observed in both cases. Reduction of the load of Vibro anguillarum on rotifers by applying herbal extracts during enrichment process has been achieved by Takaoka et al. ( 2011 ). While the above mentioned techniques are costly and labor intensive, rinsing of rotifers with clean water removes the majority of bacteria which dwells on the surfaces of the rotifers and culture water. Therefore, the current study indicated that rinsing of rotifers and culture water of B.calyciflorus reduced the bacterial population by half in all the treatments (Table 4 ). Concerning the differences in the total bacterial population with the different organic media applied, the 100% BBM and POMF substituted groups gave insignificant difference in total bacterial count in non-rinsed rotifers while the 100% BBM gave significantly lower bacterial load in rinsed rotifers. The SHMF gave significantly highest total bacterial count in the non-rinsed rotifers followed by the WHCF substituted groups. This result is in agreement with øie et al. ( 1994 ) who reported that rotifers cultured on microalgae have low microbial load compared to rotifers cultured on yeast. Overall the bacterial load of B.calyciflorus in the current study followed an inverse relationship with the microalgal culture performance in which organic media with better Chlorella sp. culture performance showed lower bacterial load in both rinsed and non-rinsed rotifers. This can be related to the nutrient competition between bacteria and microalgae where the Chlorella sp. growth is better; there will be less nutrient for the bacteria to proliferate. Therefore, the bacterial load of rinsed and non-rinsed rotifers of the SHMF and compost filtrate from the different parts of water hyacinth showed the highest counts compared to the POMF and 100% BBM groups. 5. Conclusion and recommendation From this study it can be concluded that 50% substitution of BBM by poultry manure filtrate (POMF) gave the highest Chlorella sp. growth and subsequently higher B.calyciflorus population density. POMF also gave insignificant total bacterial count with the 100% BBM control when the rotifers were not rinsed with autoclaved tap water. On the other hand, SHMF and water hyacinth compost filtrate showed the highest microbial load in both rinsed and non-rinsed rotifers. It can also be concluded that rinsing of rotifers with autoclaved tap water can be used as an economical way of reducing microbial loads by half which intern reduces the risk of opportunistic pathogen introduction into the larvae feeding on the rotifers. SHMF and water hyacinth compost filtrate gave the lowest C hlorella sp. culture and supports minimum B.calyciflorus population density. Based on the results of this study POMF may totally substitute the standard media for Chlorella sp . culture despite the limitations of this experiment to incorporate other algal quality parameters such as fatty acid profile and lipid production. Therefore it is recommended that experiments be conducted with different strengths of POMF for economically important microalgae species of the country without the synthetic media incorporated and additional feed quality parameters such as fatty acid profile and lipid content of the microalgae. It is also recommended to rinse well rotifers before feeding to fish larvae to reduce surface bacterial load if advanced disinfection possibilities are not affordable. In addition, a precise identification of the diverse mix of bacteria would be good using further microbiological analysis, such as staining, biochemical tests, or molecular techniques as the basic plate count method provides only a preliminary insights into the whole cultivable bacterial population. Organic media application in microalgae culture could not be tailored to only wastewater treatment but also production of valuable products such as live feeds for fish larval culture. Declarations Competing interests: The authors declare that they have no conflict of interest. Funding: There are no specific funding received for this experiment Author Contribution All authors contributed to the conception and design of this study. Solomon Melaku conducted the whole experiment. All authors involved in the analyses and interpretation of the results. The draft of the manuscript was written by Solomon Melaku while all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors would like to thank Dr. Demeke Kifle and Dr. Samson Tilahun for providing Laboratory space and algal culture chamber for the running of the experiments. We also would like to thank Ethiopian Biodiversity Institute for providing the algal strain for the experiment. Data availability: The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. 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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-4690614","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":334735608,"identity":"b9d54bf1-2cae-4e47-a6c4-6f268d832481","order_by":0,"name":"Solomon Melaku","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYFAC5gY4g7GBwQbC5sGrhbEBzgAy0xBacGtD1XKYsBb59sbGRzcq7jDoAhkfZ7adl+efkcD44G0bg5w9Di0GZw42G+ececZgBmRIbmy7bTjjRgKz4dw2BmNcthhIJLZJ57YdZjC7kdgg+bDtNmPDjQQ2ad42hsQeXA6bAdNy/2Hzz4dt5+zn30hg/w3UUo9LC8MNuC2MbUCHHUjcALSFGaglAafDIH45zGN2JrHNcsa55OSNZx42S845J2HYcwBXiDUffJxTcVjO7Pjhwzd7yuxs5x1PPvjhTZmNPHsDLpdBALIrwDElgV/9KBgFo2AUjAK8AACbTmFuQYUYQQAAAABJRU5ErkJggg==","orcid":"","institution":"Debre Berhan University","correspondingAuthor":true,"prefix":"","firstName":"Solomon","middleName":"","lastName":"Melaku","suffix":""},{"id":334735610,"identity":"e754d81b-e155-4999-8b52-ab0112361fca","order_by":1,"name":"Abebe Getahun","email":"","orcid":"","institution":"Addis Ababa University","correspondingAuthor":false,"prefix":"","firstName":"Abebe","middleName":"","lastName":"Getahun","suffix":""},{"id":334735612,"identity":"4b1ac273-68c0-4ec6-81c8-987a34dbcc09","order_by":2,"name":"Seyoum Mengestou","email":"","orcid":"","institution":"Addis Ababa University","correspondingAuthor":false,"prefix":"","firstName":"Seyoum","middleName":"","lastName":"Mengestou","suffix":""},{"id":334735614,"identity":"da30c2ab-0ffe-4693-bf28-fc1cb9a7d66e","order_by":3,"name":"Akewake Geremew","email":"","orcid":"","institution":"Addis Ababa University","correspondingAuthor":false,"prefix":"","firstName":"Akewake","middleName":"","lastName":"Geremew","suffix":""},{"id":334735616,"identity":"c13f5379-87fe-4d7b-9fd3-e003530e5126","order_by":4,"name":"Amha Belay","email":"","orcid":"","institution":"ALGAE4ALL, LLC Latigo Cir.La Quinta","correspondingAuthor":false,"prefix":"","firstName":"Amha","middleName":"","lastName":"Belay","suffix":""}],"badges":[],"createdAt":"2024-07-05 08:04:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4690614/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4690614/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61730694,"identity":"180d6adb-0487-4d1e-93e7-f3bc683f26fc","added_by":"auto","created_at":"2024-08-05 00:39:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49002,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing Lake Tinishu Abaya from Ethiopian lakes and the sampling sites (Shores 1 and 2 and one open water area 3)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4690614/v1/c906abee3d9664ea2ee51fbc.png"},{"id":61730520,"identity":"fbd3461c-9177-4904-a0b4-258bd32aac52","added_by":"auto","created_at":"2024-08-05 00:31:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76709,"visible":true,"origin":"","legend":"\u003cp\u003eOptical density monitoring of microalgae culture at 750nm (a) and 686nm (b). Values are mean ± SD, n=2, Where, BBM=100% Bolds Basal Medium, POMF= poultry manure filtrate, SHMF= sheep manure filtrate, WHRCF= water hyacinth root compost filtrate, WHLCF= water hyacinth leaf compost filtrate, WHR+LCF= water hyacinth root and leaf compost filtrate\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4690614/v1/e92400e61ce6b8515c6efad6.png"},{"id":61730897,"identity":"927e7a85-b092-4ef2-84d2-2ca07adc3358","added_by":"auto","created_at":"2024-08-05 00:47:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1040220,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4690614/v1/eede9d01-2206-4154-ac66-8617f7b45990.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reproduction rate and microbial load of the rotifer Brachionus calyciflorus (Pallas, 1766) fed on Chlorella sp. cultured on organic media","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eBrachionus calyciflorus\u003c/em\u003e is one of the live feed organisms used in the larviculture of freshwater finfishes (Lim \u0026amp; Wong \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Aoyama et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ekelemu \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This rotifer has been a good substitute for the costly \u003cem\u003eArtemia\u003c/em\u003e cyst import as it is locally available for most resource limited developing countries (Arimoro \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ekelemu \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Melaku et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, due to its relative small size, \u003cem\u003eB.calyciflorus\u003c/em\u003e is used to wean ornamental fish larvae with small mouth gape during their initial feeding, as they are unable to ingest the larger-sized \u003cem\u003eArtemia\u003c/em\u003e nauplii (Lim \u0026amp; Wong \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lahnsteiner et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eB.calyciflorus\u003c/em\u003e is also used successfully in the weaning of African catfish larvae in different settings (Awa\u0026iuml;ss \u0026amp; Kestemont \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Evangelista et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Abaho et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kwikiriza et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, the mass culture of \u003cem\u003eB.calyciflorus\u003c/em\u003e is hindered by a lack of suitable artificial diets to substitute the costly microalgae, which adds extra cost to the larviculture of commercially important fish species.\u003c/p\u003e \u003cp\u003eMicroalgae have been an indispensible part of rotifer culture systems providing stable and clean cultures (Lavens and Sorgeloos \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Nagappan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, the nutritional composition of rotifers cultured on microalgae has superior quality compared to those fed with artificial diets (Dhert et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). microalgae are primarily utilized in live feed culture to transfer essential nutrients, such as polyunsaturated fatty acids (PUFA) to the finfish larvae (Concei\u0026ccedil;\u0026atilde;o et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Richmond and Hu \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite their benefits, the widespread application of microalgae in aquaculture is hindered by the costly synthetic media usage and complex culture systems required (Chauton et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, alternatives to the costly synthetic media in the form of either total or partial substitution with different forms and pre-processing of organic manure resources have been an area of research recently (Asmare et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bauer et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These organic manure resources are readily available and have the potential to replace totally or partially the costly synthetic media if properly processed and applied (Bjornsson et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the other hand, application of these manure resources for microalgae culture has an implication of reducing the impacts of the resources on the environment if disposed directly. Due to this fact most applications of this manure filtrates in microalgae culture are mainly aimed at reclaimng the organic wastes to mitigate their environmental impacts and enhancing lipid production of some microalgae species for biodiesel production (Wang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pszcz\u0026oacute;łkowska et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eChlorella sp.\u003c/em\u003e, especially when grown mixotrophically and heterotrophically, demonstrate robust growth rates and high biomass productivity, which is essential for industrial applications.These species are capable of accumulating significant amounts of intracellular lipids, making them suitable for biofuel production. The lipid content can range from 20\u0026ndash;70% of cell weight, leading to high yields. The potential for nutrient recycling in heterotrophic microalgae cultures, which can reduce the demand for virgin nutrients and water, thus enhancing the sustainability of the production process. \u003cem\u003eChlorella sp.\u003c/em\u003e have been successfully used in various commercial applications, including nutritional supplements and high-value fatty acids, due to their desirable qualities (Lowrey et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile organic manure filtrates have been primarily used to reclaim organic wastes and enhance lipid production, limited research has explored their application in microalgae culture for live feed production (Dahril \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ogello et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This is partly due to biosafety concerns associated with the potential presence of pathogenic microorganisms in organic manure, which could pose risks to aquaculture species fed on live feed. High organic matter load in rotifer culture tanks, in combination with high temperature, favors bacteria to proliferate (Skjermo and Vadstein \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), some of which may be pathogenic to rotifers(Yu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Although most bacteria have no detrimental effect on rotifers themselves, the real concern lies in the risk to host (larvae) feeding on the rotifers (Skjermo and Vadstein \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, the organic manure resources could be an alternative source of nutrients for microalgae culture for live feed production and green water aquaculture systems in general with proper pre-processing and culture system combinations in resource-limited developing countries such as Ethiopia where organic manure resources are abundant (Samani \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, this study aims to evaluate the effect of substituting costly synthetic media with different organic media resources on microalgae culture performance and its subsequent effect on the culture performance of \u003cem\u003eB. calyciflorus\u003c/em\u003e as live feed for larval fish culture.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental set up\u003c/h2\u003e \u003cp\u003eThe experiment was conducted at the Phycology laboratory of the Department of Zoological Sciences of Addis Ababa University. The experiment acknowledges the inherent differences in nutrient composition of the organic media derived from different sources. This variability is expected to impact the growth performance of the microalgae and rotifers. The study attempts to standardize conditions by autoclaving the organic media to eliminate pathogens and storing them under controlled temperatures. In addition, carbon to nitrogen ratio was maintained at 24 for all treatments according to the recommendations by Gao et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) through serial dilution with distilled water before being used as a substitute to the BBM. The experiment compares the growth performance of microalgae and rotifers across different treatments, providing insights into the suitability of each organic media as a BBM substitute.\u003c/p\u003e \u003cp\u003eIn the first phase of the experiment, \u003cem\u003eChlorella sp.\u003c/em\u003e culture was conducted with a replacement of 50% of Bold\u0026rsquo;s Basal Media (BBM) with five organic media and a control with 100% standard BBM in duplicate flasks for 10 days. The experimental treatments were poultry manure filtrate (POMF), sheep manure filtrate (SHMF), water hyacinth root compost filtrate (WHRCF), water hyacinth leaf compost filtrate (WHLCF), water hyacinth root and leaf compost filtrate (WHR\u0026thinsp;+\u0026thinsp;LCF) and a control treatment with 100% BBM. At the eleventh day of the culture period, \u003cem\u003eB.calyciflorus\u003c/em\u003e rotifer was inoculated at a density of 20 rotifers mL \u003csup\u003e-1\u003c/sup\u003e to each culture flask. The rotifer culture was kept for 4 days to evaluate the capacity of the \u003cem\u003eChlorella sp.\u003c/em\u003e cultured in the different culture media on the rotifer reproduction rate. At the end of the rotifer culture period, rotifers were enumerated and compared among the different treatments for their carrying capacity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microalgae strain and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eChlorella sp.\u003c/em\u003e with accession number EMCC-M31 Lake Beseka strain was provided by the Ethiopian Biodiversity Institute, Addis Ababa, Ethiopia. The \u003cem\u003eChlorella sp.\u003c/em\u003e stock was inoculated in a 250 mL flask with 200 mL of BBM media at pH 8. The microalga was subsequently up scaled to cell densities appropriate to start the experiment. Then 50% of the Bolds\u0026rsquo; Basal Medium (BBM) was substituted with the respective organic media except for the control with 100% BBM culture. The culture was run for 10 days using 1L Erlenmeyer flasks with 800mL culture volume. The microalgae culture was run mixotrophically under a culture chamber with 27 \u0026micro;mol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e light intensity. The photo period was set at 16 to 8 light and dark periods respectively. The daily cell growth rate was monitored using optical density measurement at 750nm and 686nm (Chioccioli et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eChlorella sp.\u003c/em\u003e growth rate was calculated using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\:\\:\\:\\:\\:\\:\\:\\:{\\mu\\:}\\:=\\:\\:\\text{l}\\text{n}(n1/n2)/(t2-t1)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u0026micro; is the specific growth rate (day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), n1 and n2 are initial and final cell density counts (cells/mL), while t1 and t2 are initial and final culture periods in days. The micro algal cell counts were carried out at the start and last dates of the culture using Sedgwick rafter cells. Bold\u0026rsquo;s Basal Medium (BBM), was prepared from 10mL per liter stock solutions of 25 g L\u003csup\u003e-1\u003c/sup\u003e NaNO\u003csub\u003e3\u003c/sub\u003e, 2.5 g L\u003csup\u003e-1\u003c/sup\u003e of CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO, 7.5 g L\u003csup\u003e-1\u003c/sup\u003e of MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 7.5 g L\u003csup\u003e-1\u003c/sup\u003e of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 17.5 g L\u003csup\u003e-1\u003c/sup\u003e of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.5 g L\u003csup\u003e-1\u003c/sup\u003e of NaCl and 1 mL per liter of 50 g L\u003csup\u003e-1\u003c/sup\u003e EDTA anhydrous, 31 g L\u003csup\u003e-1\u003c/sup\u003e of KOH, 4.98 g L\u003csup\u003e-1\u003c/sup\u003e of FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 1 mL of H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 11.4 g L\u003csup\u003e-1\u003c/sup\u003e of H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 8.82 g L\u003csup\u003e-1\u003c/sup\u003e ZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 1.44 g L\u003csup\u003e-1\u003c/sup\u003e of MnCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO, 0.71 g L\u003csup\u003e-1\u003c/sup\u003e of MoO\u003csub\u003e3\u003c/sub\u003e, 1.57 g L\u003csup\u003e-1\u003c/sup\u003e of CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO, 0.49 g L\u003csup\u003e-1\u003c/sup\u003e of Co(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO. The initial pH of the medium was adjusted to 8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Organic media processing and preparation\u003c/h2\u003e \u003cp\u003ePoultry and sheep manures used in the experiment were obtained from Asko and Winget area small scale poultry and sheep markets in Addis Ababa while the water hyacinth compost filtrate were obtained from an experimental field at Batu fisheries and other aquatic life research center, Oromia Agricultural Research Institute. The samples were collected in August 2022. Organic media were dried immediately to a constant weight over sunlight and ground to a fine particulate matter using a laboratory grinder. Afterwards, 1 kg of each organic manure was soaked with 3 liter distilled water overnight. Then, the filtrates were decanted and filtered with 30\u0026micro;m sieve to a constant volume of 1 liter. Afterwards the organic media were autoclaved at 121\u003csup\u003e0\u003c/sup\u003ec for 15 minutes to remove potential pathogenic microorganisms and zooplankton. The organic media were stored in a refrigerator below \u0026minus;\u0026thinsp;4 \u003csup\u003e0\u003c/sup\u003ec until being applied to the experiments and chemical composition analysis. The chemical composition of the organic media was measured photo metrically using (APHA \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) standard methods (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the physico-chemical properties of the constituents of the culture media\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePOMF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSHMF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWHRCF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWHLCF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWHR\u0026thinsp;+\u0026thinsp;LCF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTP (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e2\u003c/sub\u003e-N (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e-N (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC:N ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTK (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSS (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e130.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e201.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e167.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTDS (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e939\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1275\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCond (\u0026micro;S/cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1804\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTurb (NTU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eWhere, POMF\u0026thinsp;=\u0026thinsp;poultry manure filtrate, SHMF\u0026thinsp;=\u0026thinsp;sheep manure filtrate, WHRCF\u0026thinsp;=\u0026thinsp;water hyacinth root compost filtrate, WHLCF\u0026thinsp;=\u0026thinsp;water hyacinth leaf compost filtrate, WHR\u0026thinsp;+\u0026thinsp;LCF\u0026thinsp;=\u0026thinsp;water hyacinth root and leaf compost filtrate\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe samples were analyzed following the standard methods described in (APHA \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). To briefly describe the procedures, TP contents of the culture media was measured by the ascorbic acid technique after digestion with persulfate. Nitrate (NO\u003csub\u003e3\u003c/sub\u003e-N) was measured with the sodium salicylate method, while ammonia (NH\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e+\u003c/sup\u003e NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e- N) was determined by the phenate method. Nitrite (NO\u003csub\u003e2\u003c/sub\u003e-N) was determined by diazotization with Sulphanilamide and coupling to Naphthylethylene diamine di-HCl. Total Nitrogen was determined by the persulfate digestion method while total Potassium concentration was measured using Tetraphenylborate method using DR6000 HACH Spectrophotometer. Total suspended solid (TSS) was determined gravimetrically after titration of a known volume of organic media sample. The COD of the samples was determined by digesting the samples in an aluminum heating block for about 7 minutes. The Carbon to Nitrogen ratio was calculated by dividing the COD values by the total nitrogen values of the respective samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Rotifer strain and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003e\u0026ldquo;Brachionus calyciflorus Pallas 1766\u0026rdquo;\u003c/em\u003e were isolated from Lake Tinishu Abaya, Gurage zone, Ethiopia (plate 1a). Lake Tinishu Abaya is situated in the rift valley system of Ethiopia at 7\u003csup\u003e0\u003c/sup\u003e29'03.65\" N latitude and 38\u003csup\u003e0\u003c/sup\u003e03'17.79\" E longitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The lake covers a surface area of 1253 hectares with maximum and mean depths of 1.5 and 1.1 m respectively (Yirga and Brook \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To briefly describe the procedures, mixed population of zooplankton samples were collected by plankton net of 15 \u0026micro;m towed horizontally over the lakes surface several times. Several liters of lake water was filtered with the plankton net to obtain significant amount of zooplankton including \u003cem\u003eB. calyciflorus\u003c/em\u003e and concentrated to a four liter sample collection bottles. Subsequent sieving of the samples was done over 300 and 600 \u0026micro;m size sieves to remove bigger zooplanktons and particulate matter. Afterwards, the samples were transported to Phycology Laboratory of the Department of Zoological Sciences, Addis Ababa University. Water physico-chemical parameters were recorded onsite with portable multi parameter probe (HACH hd401dn, Loveland, USA) (Data not presented). Up on arrival to the laboratory, confirmation of the presence of \u003cem\u003eB.calyciflorus\u003c/em\u003e was checked under compound microscope at magnifications of 10x and 20x. One ml of sample was treated with a drop of lugol\u0026rsquo;s solution for better resolution and identification of the \u003cem\u003eB.calyciflorus\u003c/em\u003e was done according to Koste (1978) (Plate 1a). Serial dilution method was used to isolate the \u003cem\u003eB.calyciflorus\u003c/em\u003e from other rotifers of nearly similar size as the population density of the \u003cem\u003eB.calyciflorus\u003c/em\u003e was dominant. The rotifer stock culture was initially conducted in filtered water brought from the lake and gradually changed to reconstituted hard water composed of the following chemicals: 96 mg NaHCO\u003csub\u003e3\u003c/sub\u003e, 60 mg CaSO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO, 60 mg MgSO\u003csub\u003e4\u003c/sub\u003e, and 4 mg KCL in one liter of distilled water according to Lavens \u0026amp; Sorgeloos (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The rotifers were cultured on \u003cem\u003eChlorella sp.\u003c/em\u003e at a density of 1x10\u003csup\u003e7\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a temperature of 25\u003csup\u003e0\u003c/sup\u003eC with a photo period of 12hr light and 12hr dark (Rico-martinez and Dodsonb \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Afterwards the rotifer cultures were up scaled subsequently until sufficient numbers were obtained to start the experiment. At the end of the four days culture, rotifers were harvested using 75 \u0026micro;m sieve and rotifer reproduction rate was calculated using the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\:\\:\\:\\:\\:\\:\\:\\:{\\mu\\:}\\:=\\:\\:\\text{l}\\text{n}(n1/n2)/(t2-t1)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u0026micro; is the specific growth rate (day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), n1 and n2 are initial and final cell density counts (cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while t1 and t2 are initial and final culture periods in days\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Total bacterial counts\u003c/h2\u003e \u003cp\u003eTwo types of samples were taken for the total bacterial counts i.e. samples taken before the rotifers were rinsed with autoclaved tap water and samples taken after rinsing the rotifers with autoclaved tap water. One milliliter sample of rotifers along with culture water was taken from each replicate Erlenmeyer flasks in both cases. The bacterial culture was conducted at Mycology laboratory of the Department of Zoological Sciences, Addis Ababa University. Serial dilutions of the samples were made by transferring 0.1 mL of experimental samples to 0.9 mL of sterile freshwater and a pretest was conducted to determine the countable ranges before the whole samples were seeded on the petri plates. Afterwards, the appropriate dilutions were plated on Tryptic Soy Agar (TSA, HiMedia, India) in duplicates and incubated for 48 hrs at 37\u003csup\u003e0\u003c/sup\u003ec. After 48 hrs incubation, plates with distinct colonies in the range of 30 to 400 colony forming units were counted and the results were expressed as CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (plate 2). All these procedures were carried out under sterile conditions to prevent contamination and to obtain accurate results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Water quality parameters\u003c/h2\u003e \u003cp\u003eCulture water temperature, dissolved oxygen, pH, total ammonia nitrogen, nitrite and nitrate were measured daily during the four day rotifer culture. Portable pH and DO meters were used to measure the respective parameters \u003cem\u003ein-situ\u003c/em\u003e. Colorimetric method was applied to measure the concentration of TAN, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N in mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using API freshwater master test kit (API\u0026reg;, Pennsylvania, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data analysis\u003c/h2\u003e \u003cp\u003eAll the experimental data were analyzed using OriginPro learning edition release 2024 (Originlab, USA). Data were evaluated for homogeneity of variances using Levene's test. When the data were found to follow normal distribution and to be homoscedastic, final day rotifer densities, micro algal cell densities, micro algal growth rate and total bacterial counts of rinsed and non-rinsed rotifers were compared between treatments using one-way analysis of variance (ANOVA), followed by Tukey HSD test for multiple comparisons of means. However, when these assumptions were not fulfilled, Kruskal - Wallis rank Sum test was applied to compare means.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effects of partial substitution of BBM with organic media on the growth of \u003cem\u003eChlorella sp.\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe results of this experiment indicated that 50% substitution of BBM by POMF gave the highest final day cell density followed by the control with 100% BBM, but there was no significant difference ( p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in algal growth rate between the two groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). SHMF substituted cultures gave the third highest \u003cem\u003eChlorella sp.\u003c/em\u003e growth in terms of cell density at the last date of the culture period. On the other hand, the three treatment groups with water hyacinth-based compost filtrate gave significantly the lowest \u003cem\u003eChlorella sp.\u003c/em\u003e cell density and growth rate compared to the POMF and BBM culture groups. There was no significant difference in cell density between the three water hyacinth-based compost filtrates that substituted 50% BBM.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eChlorella sp.\u003c/em\u003e growth on organic media substituted cultures\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial cell density (cells/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal cell density (cells/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrowth rate (Day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.74x10\u003csup\u003e8\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44x10\u003csup\u003e6b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePOMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.83x10\u003csup\u003e8\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4.03x10\u003csup\u003e6a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0032\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.94x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65x10\u003csup\u003e6c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0032\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHRCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.66x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5.23x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.49x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14x10\u003csup\u003e5d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0012\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHLCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.25x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5.09x10\u003csup\u003e4d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0012\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHR\u0026thinsp;+\u0026thinsp;LCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.64x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.37x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82x10\u003csup\u003e4d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0007\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;2, values with different superscript letters in the same column are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tucky HD test. Where, BBM\u0026thinsp;=\u0026thinsp;100% Bolds Basal Medium, POMF\u0026thinsp;=\u0026thinsp;poultry manure filtrate, SHMF\u0026thinsp;=\u0026thinsp;sheep manure filtrate, WHRCF\u0026thinsp;=\u0026thinsp;water hyacinth root compost filtrate, WHLCF\u0026thinsp;=\u0026thinsp;water hyacinth leaf compost filtrate, WHR\u0026thinsp;+\u0026thinsp;LCF\u0026thinsp;=\u0026thinsp;water hyacinth root and leaf compost filtrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effects of \u003cem\u003eChlorella sp.\u003c/em\u003e cultured on different organic media on the population growth of \u003cem\u003eB.calyciflorus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003ePOMF substituted cultures of the microalgae gave significantly higher rotifer reproduction at the last date of the rotifer batch culture (272\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 individuals mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) followed by the 100% BBM (122.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.53 individuals mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) culture (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On the other hand, SHMF (83.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.78 individuals mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) gave the third highest rotifer reproduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The three treatments with the different water hyacinth-based composts gave the lowest rotifer reproduction corresponding with the lowest \u003cem\u003eChlorella sp\u003c/em\u003e. growth performance mentioned in section \u003cspan refid=\"Sec11\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of \u003cem\u003eChlorella sp.\u003c/em\u003e cultured on different organic media on the population growth of \u003cem\u003eB.calyciflorus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial rotifer density (individuals mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal rotifer density (individuals mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRotifer growth rate (Day \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e122.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePOMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e272\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.78\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHRCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHLCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHR\u0026thinsp;+\u0026thinsp;LCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;2, values with different superscript letters in the same column are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tucky HSD test. Where, BBM\u0026thinsp;=\u0026thinsp;100% Bolds Basal Medium, POMF\u0026thinsp;=\u0026thinsp;poultry manure filtrate, SHMF\u0026thinsp;=\u0026thinsp;sheep manure filtrate, WHRCF\u0026thinsp;=\u0026thinsp;water hyacinth root compost filtrate, WHLCF\u0026thinsp;=\u0026thinsp;water hyacinth leaf compost filtrate, WHR\u0026thinsp;+\u0026thinsp;LCF\u0026thinsp;=\u0026thinsp;water hyacinth root and leaf compost filtrate\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Microbial loads of \u003cem\u003eB.calyciflorus\u003c/em\u003e fed on \u003cem\u003eChlorella sp.\u003c/em\u003e cultured with organic media substitution\u003c/h2\u003e \u003cp\u003eThe total bacterial counts of the non-rinsed rotifers significantly differed between treatments. The total viable bacterial load of non-rinsed rotifer cultures with POMF (8.05x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94x10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e-1\u003c/sup\u003e) and 100% BBM (6.9x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9x10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e-1\u003c/sup\u003e) was significantly lower compared to the other groups while the SHMF (1.62x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83x10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e-1\u003c/sup\u003e) substituted culture was significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The water hyacinth compost filtrate substituted groups gave also higher total viable bacterial counts compared to the POMF and 100% BBM culture but lower as compared to the SHMF substituted groups (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, plate 2).\u003c/p\u003e \u003cp\u003eThe total viable bacterial count of rinsed rotifers also showed significant difference amongst the different treatments and the control as well. To this end, the 100% BBM gave significantly lowest total bacterial count (7.95x10\u003csup\u003e3\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.19x10\u003csup\u003e2\u003c/sup\u003e CFU mL\u003csup\u003e-1\u003c/sup\u003e) of rinsed rotifers compared to the organic media substituted groups. On the other hand, the WHR\u0026thinsp;+\u0026thinsp;LCF gave significantly higher total bacterial count of rinsed rotifers compared to the other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eTable 4. Microbial loads of \u003cem\u003eB.calyciflorus\u003c/em\u003e fed on \u003cem\u003eChlorella sp.\u003c/em\u003e cultured with substitution of BBM with organic media.\u0026nbsp;\u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal bacterial count\u003c/p\u003e \u003cp\u003e(Non-rinsed) (CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal bacterial load\u003c/p\u003e \u003cp\u003e(Rinsed) (CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.9x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9x10\u003csup\u003e5d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.95x10\u003csup\u003e3\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.19x10\u003csup\u003e2c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePOMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.05x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94x10\u003csup\u003e5cd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.44x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63x10\u003csup\u003e3b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSHMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.62x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83x10\u003csup\u003e5a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.82x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19x10\u003csup\u003e3ab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHRCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.09x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7.78x10\u003csup\u003e5b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.63x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.89x10\u003csup\u003e2ab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHLCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7.07x10\u003csup\u003e4b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.92x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13x10\u003csup\u003e3ab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHR\u0026thinsp;+\u0026thinsp;LCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.03x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7.07x10\u003csup\u003e4bc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.05x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;8.48x10\u003csup\u003e2a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;2, values with different superscript letters in the same column are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tucky HSD test. Where, BBM\u0026thinsp;=\u0026thinsp;100% Bolds Basal Medium, POMF\u0026thinsp;=\u0026thinsp;poultry manure filtrate, SHMF\u0026thinsp;=\u0026thinsp;sheep manure filtrate, WHRCF\u0026thinsp;=\u0026thinsp;water hyacinth root compost filtrate, WHLCF\u0026thinsp;=\u0026thinsp;water hyacinth leaf compost filtrate, WHR\u0026thinsp;+\u0026thinsp;LCF\u0026thinsp;=\u0026thinsp;water hyacinth root and leaf compost filtrate\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Water quality parameters\u003c/h2\u003e \u003cp\u003eBasic water quality parameters such as temperature, pH, DO and nitrogenous compounds (TAN, NO\u003csub\u003e2\u003c/sub\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e-N) were measured daily during the four day rotifer culture whereas the measured values at the last date of the culture period are presented in Table \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWater quality parameters measured at the final date of rotifer culture\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWHRCF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWHLCF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWHR\u0026thinsp;+\u0026thinsp;LCF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePOMF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSHMF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBBM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature (0\u003csup\u003ec\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e8.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDO (mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTAN (mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (mg L\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eValues are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;2, Where, BBM\u0026thinsp;=\u0026thinsp;100% Bolds Basal Medium, POMF\u0026thinsp;=\u0026thinsp;poultry manure filtrate, SHMF\u0026thinsp;=\u0026thinsp;sheep manure filtrate, WHRCF\u0026thinsp;=\u0026thinsp;water hyacinth root compost filtrate, WHLCF\u0026thinsp;=\u0026thinsp;water hyacinth leaf compost filtrate, WHR\u0026thinsp;+\u0026thinsp;LCF\u0026thinsp;=\u0026thinsp;water hyacinth root and leaf compost filtrate\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e4.1 Chlorella sp\u003c/em\u003e. culture performance on different organic media substitutions\u003c/h2\u003e \u003cp\u003eThe culture performance in terms of culture density, growth rate and optical density measurement at the final day of the culture period indicated that POMF-substituted cultures achieved significantly higher cell density compared to other treatments, followed by the 100% BBM. This result is in agreement with the findings of Markou et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) where \u003cem\u003eChlorella vulgaris\u003c/em\u003e showed significantly higher biomass production on raw poultry litter leachate-based media compared to the standard BG-11 media. This might be partly due to the ability of the \u003cem\u003eChlorella sp\u003c/em\u003e. to grow mixotrophicaly which is an attribute of the species to grow in light limited, murky media which most microalgae cannot tolerate (Heredia-Arroyo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bansal, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). One critical challenge in using organic manure as a culture medium for microalgae is the high amount of suspended solids that blocks light penetration, a critical component in algae culture setups (Wang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, the notable effect of supporting higher growth of \u003cem\u003eChlorella sp.\u003c/em\u003e can be attributed to the relatively higher levels of major nutrients such as Phosphorus and Potassium in the poultry manure filtrate compared to other treatments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Agwa \u0026amp; Abu (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) found that \u003cem\u003eChlorella sp.\u003c/em\u003e cultured in poultry manure medium showed higher lipid production, achieving nearly 18% (w/w) lipid content under sunlight as light source. Rajagopal et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that \u003cem\u003eChlorella vulgaris\u003c/em\u003e was able to grow and utilize nutrients from a 10% diluted chicken manure digestate with better cell growth rate and biomass productivity. Another study by Dincă et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) indicated that \u003cem\u003eChlorella spp\u003c/em\u003e. produced higher biomass compared to the BG-11 standard media.\u003c/p\u003e \u003cp\u003eOn the other hand, the sheep manure filtrate gave the third highest microalgal cell density despite its nutrient composition competing with the poultry manure filtrate and 100% BBM control. This might be attributed to the heavily dark coloration of the SHMF which inhibited complete light blockage which shifts the mode of microalgae culture from mixotrophic which \u003cem\u003eChlorella sp.\u003c/em\u003e performed better to heterotrophic culture mode (Liang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mohammad Mirzaie et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, there were bubble formations with the SHMF culture setups during aeration which might affect the growth of the microalgae (plate 1c left end). Three treatments with the different parts of water hyacinth compost filtrate gave the least \u003cem\u003eChlorella sp.\u003c/em\u003e growth as indicated in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This is partly due to the suboptimal nutrient composition compared to the other treatments and the heavy dark coloration of the organic media even worse than the SHMF as mentioned above (plate 1c right). Although Water hyacinth compost application is more commonly reported in soil amendment in crop production (Malik \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sagar et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), few studies report its application in microalgae culture (Iba et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dahiya et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Iba et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that \u003cem\u003eChlorella vulgaris\u003c/em\u003e cultured at 5% water hyacinth liquid fertilizer gave better cell density, specific growth rate and biomass production in 40 mg L\u003csup\u003e-1\u003c/sup\u003e salinity level. Conversely, Dahiya et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that water hyacinth hydrolysate, combined with inorganic carbon and a 12:12 hour light-dark photoperiod, produced the highest biomass of nearly 200 mg/L in two strains of \u003cem\u003eChlorella sorokiniana\u003c/em\u003e isolated from municipal wastewater.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2 B.calyciflorus\u003c/b\u003e \u003cb\u003eculture performance on the\u003c/b\u003e \u003cb\u003eChlorella sp.\u003c/b\u003e \u003cb\u003ecultured under substitution of algal growth media with different organic media\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eB.calyciflorus\u003c/em\u003e culture performance in terms of rotifer growth rate and population density at the last date of the culture period followed the same trend of the algal culture performance. The \u003cem\u003eChlorella sp.\u003c/em\u003e Culture with POMF substituted group gave significantly higher \u003cem\u003eB. calyciflorus\u003c/em\u003e growth rate and population density followed by the 100% BBM culture. This result is in agreement to the reports of Dahril (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) in which chicken manure gave the highest cell density of \u003cem\u003eChlorella spp.\u003c/em\u003e and subsequently supported higher \u003cem\u003eB.calyciflorus\u003c/em\u003e population compared to duck, quail, buffalo and horse manures. The author suggested that high nitrogen content of the chicken manure medium contributed to this effect. In another study by Ogello \u0026amp; Hagiwara, (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) chicken manure extract at 2 mL L\u003csup\u003e-1\u003c/sup\u003e supplied simultaneously with \u003cem\u003eChlorella vulgaris\u003c/em\u003e in the culture jars gave significantly higher \u003cem\u003eB.calyciflorus\u003c/em\u003e population growth rate and population density. A more recent study by Zhu et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) indicated that 10mL L\u003csup\u003e-1\u003c/sup\u003e of chicken manure extract cultured \u003cem\u003eChlorella vulgaris\u003c/em\u003e promoted the growth rate and changed the life history of the rotifer \u003cem\u003eBrachionus plicatilis\u003c/em\u003e compared to the standard BG-11 medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Bacterial load of \u003cem\u003eB.calyciflorus\u003c/em\u003e reared on \u003cem\u003eChlorella sp.\u003c/em\u003e cultured on different organic media\u003c/h2\u003e \u003cp\u003eThe main challenges in the application of organic manure resources in live feed culture setups is the potential risk of introducing pathogenic microorganisms into the culture system and to the fish larvae feeding on the live feeds. Rotifer culture systems are characterized with high organic matter load and when this organic matter load is accompanied by high culture temperature, the resultant effect creates high bacterial load in the culture water and rotifers. Since rotifers are filter feeders which can ingest bacteria, the bacterial community of rotifers is similar to the bacterial community of the culture water (Starkweather et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Seaman et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Skjermo and Vadstein \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Rotifers were found to be the major vectors of pathogenic bacteria for fish larvae in previous studies (Verdonck et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, decontamination of organic manure resources through different techniques is crucial from the biosecurity point of view. To this end, the organic manure resources applied in this study were autoclaved at 121\u003csup\u003e0\u003c/sup\u003ec for 15 minutes before being applied to the \u003cem\u003eChlorella sp.\u003c/em\u003e culture and its application for \u003cem\u003eB.calyciflorus\u003c/em\u003e culture. Afterwards, the bacterial load of \u003cem\u003eB.calyciflorus\u003c/em\u003e was evaluated using the classical culture based method in rinsed and non-rinsed rotifers with an autoclaved tap water. Use of sodium nifurstyrenate with bacterial reduction rate of 10 to 100 times have been reported by Tanasomwang \u0026amp; Muroga (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) where the microbial community composition shifted to less \u003cem\u003eVibrio\u003c/em\u003e, but higher \u003cem\u003ePseudomonas.\u003c/em\u003e Munro et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) found that UV light could reduce the bacterial load by more than 90% and Suantika et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) used ozone in a rotifer recirculation system to reduce bacterial densities. However, microbial composition changes were not observed in both cases. Reduction of the load of \u003cem\u003eVibro anguillarum\u003c/em\u003e on rotifers by applying herbal extracts during enrichment process has been achieved by Takaoka et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). While the above mentioned techniques are costly and labor intensive, rinsing of rotifers with clean water removes the majority of bacteria which dwells on the surfaces of the rotifers and culture water. Therefore, the current study indicated that rinsing of rotifers and culture water of \u003cem\u003eB.calyciflorus\u003c/em\u003e reduced the bacterial population by half in all the treatments (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConcerning the differences in the total bacterial population with the different organic media applied, the 100% BBM and POMF substituted groups gave insignificant difference in total bacterial count in non-rinsed rotifers while the 100% BBM gave significantly lower bacterial load in rinsed rotifers. The SHMF gave significantly highest total bacterial count in the non-rinsed rotifers followed by the WHCF substituted groups. This result is in agreement with \u0026oslash;ie et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) who reported that rotifers cultured on microalgae have low microbial load compared to rotifers cultured on yeast. Overall the bacterial load of \u003cem\u003eB.calyciflorus\u003c/em\u003e in the current study followed an inverse relationship with the microalgal culture performance in which organic media with better \u003cem\u003eChlorella sp.\u003c/em\u003e culture performance showed lower bacterial load in both rinsed and non-rinsed rotifers. This can be related to the nutrient competition between bacteria and microalgae where the \u003cem\u003eChlorella sp.\u003c/em\u003e growth is better; there will be less nutrient for the bacteria to proliferate. Therefore, the bacterial load of rinsed and non-rinsed rotifers of the SHMF and compost filtrate from the different parts of water hyacinth showed the highest counts compared to the POMF and 100% BBM groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion and recommendation","content":"\u003cp\u003eFrom this study it can be concluded that 50% substitution of BBM by poultry manure filtrate (POMF) gave the highest \u003cem\u003eChlorella sp.\u003c/em\u003e growth and subsequently higher \u003cem\u003eB.calyciflorus\u003c/em\u003e population density. POMF also gave insignificant total bacterial count with the 100% BBM control when the rotifers were not rinsed with autoclaved tap water. On the other hand, SHMF and water hyacinth compost filtrate showed the highest microbial load in both rinsed and non-rinsed rotifers. It can also be concluded that rinsing of rotifers with autoclaved tap water can be used as an economical way of reducing microbial loads by half which intern reduces the risk of opportunistic pathogen introduction into the larvae feeding on the rotifers. SHMF and water hyacinth compost filtrate gave the lowest C\u003cem\u003ehlorella sp.\u003c/em\u003e culture and supports minimum \u003cem\u003eB.calyciflorus\u003c/em\u003e population density.\u003c/p\u003e \u003cp\u003eBased on the results of this study POMF may totally substitute the standard media for \u003cem\u003eChlorella sp\u003c/em\u003e. culture despite the limitations of this experiment to incorporate other algal quality parameters such as fatty acid profile and lipid production. Therefore it is recommended that experiments be conducted with different strengths of POMF for economically important microalgae species of the country without the synthetic media incorporated and additional feed quality parameters such as fatty acid profile and lipid content of the microalgae. It is also recommended to rinse well rotifers before feeding to fish larvae to reduce surface bacterial load if advanced disinfection possibilities are not affordable. In addition, a precise identification of the diverse mix of bacteria would be good using further microbiological analysis, such as staining, biochemical tests, or molecular techniques as the basic plate count method provides only a preliminary insights into the whole cultivable bacterial population. Organic media application in microalgae culture could not be tailored to only wastewater treatment but also production of valuable products such as live feeds for fish larval culture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThere are no specific funding received for this experiment\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the conception and design of this study. Solomon Melaku conducted the whole experiment. All authors involved in the analyses and interpretation of the results. The draft of the manuscript was written by Solomon Melaku while all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Dr. Demeke Kifle and Dr. Samson Tilahun for providing Laboratory space and algal culture chamber for the running of the experiments. We also would like to thank Ethiopian Biodiversity Institute for providing the algal strain for the experiment.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbaho I, Bwanika G, Walekhwa P, et al (2016) Fatty acid profiles and growth of African catfish (\u003cem\u003eClarias gariepinus\u003c/em\u003e, Burchell, 1822) larvae fed on freshwater rotifer (\u003cem\u003eBrachionus calyciflorus\u003c/em\u003e) and \u003cem\u003eArtemia\u003c/em\u003e as live starter feeds. Int J Fish Aquat Stud 4:189\u0026ndash;196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgwa OK, Abu GO (2014) Utilization of poultry waste for the cultivation of \u003cem\u003eChlorella sp\u003c/em\u003e. for biomass and lipid production. 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Asian Fish Sci 11:193\u0026ndash;201\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhert P, Rombaut G, Suantika G, Sorgeloos P (2001) Advancement of rotifer culture and manipulation techniques in Europe. In: Aquaculture. pp 129\u0026ndash;146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDincă Z, Hoaghia MA, T\u0026ouml;r\u0026ouml;k AI, et al (2021) Nutrient and organic matter removal from chicken manure leachate using \u003cem\u003eChlorella spp\u003c/em\u003e. Stud Univ Babes-Bolyai Chem 66:213\u0026ndash;220\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEkelemu JK (2015) Isolation and Mass Culture of Freshwater Rotifer (\u003cem\u003eBranchionus calyciflorus\u003c/em\u003e) Using Different Organic Media. 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Bioresour Technol 101:2623\u0026ndash;2628\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe WO, Alam S, Ostrowski AD, et al (2016) Live prey enrichment and artificial microdiets for larviculture of Atlantic red porgy Pagrus pagrus. Aquac Reports 3:93\u0026ndash;107\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan DC, Dong SL, Huang J, Zhang JS (2007) White spot syndrome virus (WSSV) transmission from rotifer inoculum to crayfish. J Invertebr Pathol 94:144\u0026ndash;148\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYirga E, Brook L (2018) Zooplankton communities as an indicator of ecosystem productivity in Lake Tinishu Abaya, Rift Valley, Ethiopia. Int J Fish Aquac 10:53\u0026ndash;70\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu JP, Hino A, Noguchi T, Wakabayashi H (1990) Toxicity of Vibrio alginolyticus on the Survival of the Rotifer \u003cem\u003eBrachionus plicatilis\u003c/em\u003e. Nippon Suisan Gakkaishi 56:1455\u0026ndash;1460\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Wu Y, Pan J, et al (2019) Pretreatment of pig manure liquid digestate for microalgae cultivation via innovative flocculation-biological contact oxidation approach. Sci Total Environ 694:133720\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu T, Jia H, Zhang H, et al (2024) Effects of chicken manure extract on the directed cultivation of bio-bait \u003cem\u003eChlorella vulgaris\u003c/em\u003e-rotifer (\u003cem\u003eBrachionus plicatilis\u003c/em\u003e) and their fatty acid content. Aquaculture 581:740387\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Plates","content":"\u003cp\u003ePlate 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Live feed, Microbial load, Organic media, Rotifer culture","lastPublishedDoi":"10.21203/rs.3.rs-4690614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4690614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn experiment was conducted to evaluate the culture performance and total microbial load of the rotifer \u003cem\u003eBrachionus calyciflorus\u003c/em\u003e reared on microalgae \u003cem\u003eChlorella sp\u003c/em\u003e. cultured with a replacement of 50% of the standard Bolds\u0026rsquo; Basal Medium (BBM) with poultry manure filtrate (POMF), sheep manure filtrate (SHMF), water hyacinth root compost filtrate (WHRCF), water hyacinth leaf compost filtrate (WHLCF), water hyacinth root and leaf compost filtrate (WHR\u0026thinsp;+\u0026thinsp;LCF) and a control treatment with 100% BBM. The results indicated that the POMF substituted culture of \u003cem\u003eChlorella sp\u003c/em\u003e. gave significantly highest cell density (1.83x10\u003csup\u003e8\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) followed by the 100% BBM culture (1.74x10\u003csup\u003e8\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) supporting 272\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 and 122.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.53 rotifers mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003eB. calyciflorus\u003c/em\u003e population, respectively. The SHMF culture gave the third highest \u003cem\u003eChlorella sp.\u003c/em\u003e density (5.94x10\u003csup\u003e7\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and supports 83.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.78 rotifers mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003eB. calyciflorus\u003c/em\u003e population. The three treatments with the compost filtrate of the different parts of water hyacinth gave the least \u003cem\u003eChlorella sp\u003c/em\u003e. cell density and \u003cem\u003eB.calyciflorus\u003c/em\u003e population as well. In terms of the total viable bacterial count of \u003cem\u003eB.calyciflorus\u003c/em\u003e cultured on the substitution of the different organic media indicated that \u003cem\u003eB.calyciflorus\u003c/em\u003e cultured on 100% BBM and 50% POMF- based culture gave significantly lowest total bacterial counts of 6.9x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9x10\u003csup\u003e5\u003c/sup\u003e and 8.05x10\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94x10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively while SHMF, WHR\u0026thinsp;+\u0026thinsp;LCF, WHLCF, WHRCF group showed higher total bacterial counts of 1.62x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83x10\u003csup\u003e5\u003c/sup\u003e, 1.09x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7.78x10\u003csup\u003e5\u003c/sup\u003e, 1.25x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7.07x10\u003csup\u003e4\u003c/sup\u003e, 1.03x10\u003csup\u003e7\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;7.07x10\u003csup\u003e4\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively in non-rinsed samples. Therefore, it can be concluded that 50% substitution the standard BBM media for \u003cem\u003eChlorella sp.\u003c/em\u003e culture with organic manures such as POMF can be a sustainable alternative to culture the rotifer \u003cem\u003eB. calyciflorus\u003c/em\u003e for larval fish culture.\u003c/p\u003e","manuscriptTitle":"Reproduction rate and microbial load of the rotifer Brachionus calyciflorus (Pallas, 1766) fed on Chlorella sp. cultured on organic media","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 00:31:03","doi":"10.21203/rs.3.rs-4690614/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-29T12:14:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-27T19:24:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-22T17:28:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148732539182168855825086839108693516030","date":"2024-09-07T09:47:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153246821613918077835315391449474734212","date":"2024-09-06T11:17:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-04T11:14:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-04T10:56:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-03T09:18:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2024-07-05T08:03:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6789494b-a479-4b1e-95d1-ea4ccdc3a72c","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-11-29T10:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-05 00:31:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4690614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4690614","identity":"rs-4690614","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}