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

doi:10.21203/rs.3.rs-4690614/v1

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

https://doi.org/10.21203/rs.3.rs-4690614/v1

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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 (1.83x10⁸ cells mL^− 1) followed by the 100% BBM culture (1.74x10⁸ 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 (5.94x10⁷ 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 6.9x10⁶ ± 9.9x10⁵ and 8.05x10⁶ ± 4.94x10⁵ CFU mL^− 1, respectively while SHMF, WHR + LCF, WHLCF, WHRCF group showed higher total bacterial counts of 1.62x10⁷ ± 2.83x10⁵, 1.09x10⁷ ± 7.78x10⁵, 1.25x10⁷ ± 7.07x10⁴, 1.03x10⁷ ± 7.07x10⁴ 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

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.

2.1 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. 1a,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 NaNO₃, 2.5 g L^-1 of CaCl₂.2H₂O, 7.5 g L^-1 of MgSO₄.7H₂O, 7.5 g L^-1 of K₂HPO₄, 17.5 g L^-1 of KH₂PO₄, 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 FeSO₄.7H₂O, 1 mL of H₂SO₄, 11.4 g L^-1 of H₃BO₃, 8.82 g L^-1 ZnSO₄.7H₂O, 1.44 g L^-1 of MnCl₂.4H₂O, 0.71 g L^-1 of MoO₃, 1.57 g L^-1 of CuSO₄.5H₂O, 0.49 g L^-1 of Co(NO₃)₂.6H₂O. 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 121⁰c for 15 minutes to remove potential pathogenic microorganisms and zooplankton. The organic media were stored in a refrigerator below − 4 ⁰c 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
NH₃-N (mg/L)	1.60	1.37	1.32	0.63	0.76
NO₂-N (mg/L)	0.10	0.13	0.20	0.13	0.16
NO₃-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 (NO₃-N) was measured with the sodium salicylate method, while ammonia (NH₃ ⁺ NH₄⁺- N) was determined by the phenate method. Nitrite (NO₂-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 7⁰29'03.65" N latitude and 38⁰03'17.79" 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 NaHCO₃, 60 mg CaSO₄.2H₂O, 60 mg MgSO₄, 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 1x10⁷ cells mL^− 1 and a temperature of 25⁰C 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 37⁰c. 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, NO₂⁺-N and NO₃⁺-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.

3.1 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	2.63x10⁶ ± 1.55x10⁴	1.74x10⁸ ± 1.44x10^6b	0.42 ± 0.0014^a	P < 0.0001
POMF	2.63x10⁶ ± 2.69x10⁴	1.83x10⁸ ± 4.03x10^6a	0.42 ± 0.0032^a
SHMF	2.63x10⁶ ± 1.13x10⁴	5.94x10⁷ ± 1.65x10^6c	0.31 ± 0.0032^b
WHRCF	2.66x10⁶ ± 5.23x10⁴	1.49x10⁷ ± 1.14x10^5d	0.17 ± 0.0012^c
WHLCF	2.63x10⁶ ± 1.98x10⁴	1.25x10⁷ ± 5.09x10^4d	0.15 ± 0.0012^d
WHR + LCF	2.64x10⁶ ± 3.25x10⁴	8.37x10⁷ ± 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 (8.05x10⁶ ± 4.94x10⁵ CFU mL^-1) and 100% BBM (6.9x10⁶ ± 9.9x10⁵ CFU mL^-1) was significantly lower compared to the other groups while the SHMF (1.62x10⁷ ± 2.83x10⁵ 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 (7.95x10³ ± 9.19x10² 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	6.9x10⁶ ± 9.9x10^5d	7.95x10³ ± 9.19x10^2c	P < 0.0001
POMF	8.05x10⁶ ± 4.94x10^5cd	2.44x10⁴ ± 1.63x10^3b
SHMF	1.62x10⁷ ± 2.83x10^5a	2.82x10⁴ ± 2.19x10^3ab
WHRCF	1.09x10⁷ ± 7.78x10^5b	2.63x10⁴ ± 9.89x10^2ab
WHLCF	1.25x10⁷ ± 7.07x10^4b	2.92x10⁴ ± 1.13x10^3ab
WHR + LCF	1.03x10⁷ ± 7.07x10^4bc	3.05x10⁴ ± 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, NO₂-N and NO₃-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
NO₂⁺-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
NO₃⁺-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

4.1 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 121⁰c 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.

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 Chlorella 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.

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.

Abaho I, Bwanika G, Walekhwa P, et al (2016) Fatty acid profiles and growth of African catfish (Clarias gariepinus, Burchell, 1822) larvae fed on freshwater rotifer (Brachionus calyciflorus) and Artemia as live starter feeds. Int J Fish Aquat Stud 4:189–196
Agwa OK, Abu GO (2014) Utilization of poultry waste for the cultivation of Chlorella sp. for biomass and lipid production. Int J Curr Microbiol Appl Sci 3:1036–1047
Aoyama Y, Moriya N, Tanaka S, et al (2015) A novel method for rearing zebrafish by using freshwater rotifers (Brachionus calyciflorus). Zebrafish 12:288–295
APHA (1995) American Public Health Association - Standard methods for the examination of water and wastewater
Arimoro FO (2006) Culture of the freshwater rotifer, Brachionus calyciflorus, and its application in fish larviculture technology. African J Biotechnol 5:536–541
Asmare AM, Demessie BA, Murthy GS (2014) Investigation of microalgae co-cultures for nutrient recovery and algal biomass production from dairy manure. Appl Eng Agric 30:335–342
Awaïss A, Kestemont P (1998) Feeding sequences (rotifer and dry diet), survival, growth and biochemical composition of African catfish, Clarias gariepinus Burchell (Pisces: Clariidae), larvae. Aquac Res 29:731–741
Bansal S (2019) Mixotrophic Growth of Chlorella Sp. Using Glycerol for the Production of Biodiesel: A Review. Mapana - J Sci 18:1–12
Bauer L, Ranglová K, Masojídek J, et al (2021) Digestate as sustainable nutrient source for microalgae—challenges and prospects. Appl. Sci. 11:1–21
Bjornsson WJ, Nicol RW, Dickinson KE, McGinn PJ (2013) Anaerobic digestates are useful nutrient sources for microalgae cultivation: Functional coupling of energy and biomass production. J Appl Phycol 25:1523–1528
Chauton MS, Reitan KI, Norsker NH, et al (2015) A techno-economic analysis of industrial production of marine microalgae as a source of EPA and DHA-rich raw material for aquafeed: Research challenges and possibilities. Aquaculture 436:95–103
Chioccioli M, Hankamer B, Ross IL (2014) Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris. PLoS One 9:1–12
Conceição LEC, Yúfera M, Makridis P, et al (2010) Live feeds for early stages of fish rearing. Aquac. Res. 41:613–640
Dahiya S, Chowdhury R, Tao W, Kumar P (2021) Biomass and lipid productivity by two algal strains of Chlorella sorokiniana grown in hydrolysate of water hyacinth. Energies 14:1–21
Dahril T (1998) Effect of Various Animal Manures on the Growth of a Freshwater Green Algae (Chlorella spp.) and Rotifer (Brachionus calyciflorus). Asian Fish Sci 11:193–201
Dhert P, Rombaut G, Suantika G, Sorgeloos P (2001) Advancement of rotifer culture and manipulation techniques in Europe. In: Aquaculture. pp 129–146
Dincă Z, Hoaghia MA, Török AI, et al (2021) Nutrient and organic matter removal from chicken manure leachate using Chlorella spp. Stud Univ Babes-Bolyai Chem 66:213–220
Ekelemu JK (2015) Isolation and Mass Culture of Freshwater Rotifer (Branchionus calyciflorus) Using Different Organic Media. Int J Adv Biol Res 5:67–72
Evangelista AD, Fortes NR, Santiago CB (2005) Comparison of some live organisms and artificial diet as feed for Asian catfish Clarias macrocephalus (Günther) larvae. J Appl Ichthyol 21:437–443
Gao F, Yang HL, Li C, et al (2019) Effect of organic carbon to nitrogen ratio in wastewater on growth, nutrient uptake and lipid accumulation of a mixotrophic microalgae Chlorella sp. Bioresour Technol 282:
Heredia-Arroyo T, Wei W, Ruan R, Hu B (2011) Mixotrophic cultivation of Chlorella vulgaris and its potential application for the oil accumulation from non-sugar materials. Biomass and Bioenergy 35:2245–2253
Iba W, Utami C, Balubi M (2019) The Growth of Chlorella vulgaris Cultured in Liquid Organic Fertilizer of Water Hyacint (Eichhornia crassipes) at Different Salinities. Aquac Indones 20:117
Kwikiriza G, Tibihika PD, Barekye A, et al (2016) Performance of African Catfish Clarias gariepinus (Clarridae) fry fed on live rotifers ( Brachionus calyciflorus ), formulated diet and a mixture of rotifers and formulated diet. Int J Fish Aquat Stud 4:11–15
Lahnsteiner F, Lahnsteiner E, Duenser A (2023) Suitability of Different Live Feed for First Feeding of Freshwater Fish Larvae. Aquac J 3:107–120
Lavens P, Sorgeloos P (1996) Manual on the production and use of live food for aquaculture. In: Fao Fisheries Technical Paper
Liang Y, Sarkany N, Cui Y (2009) Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol Lett 31:1043–1049
Lim LC, Dhert P, Sorgeloos P (2003) Recent developments in the application of live feeds in the freshwater ornamental fish culture. Aquaculture 227:319–331
Lim LC, Wong CC (1997) Use of the rotifer, Brachionus calyciflorus Pallas, in freshwater ornamental fish larviculture. Hydrobiologia 358:269–273
Lowrey J, Armenta RE, Brooks MS (2016) Nutrient and media recycling in heterotrophic microalgae cultures. Appl. Microbiol. Biotechnol. 100
Malik A (2007) Environmental challenge vis a vis opportunity: The case of water hyacinth. Environ. Int. 33:122–138
Markou G, Iconomou D, Muylaert K (2016) Applying raw poultry litter leachate for the cultivation of Arthrospira platensis and Chlorella vulgaris. Algal Res 13:79–84
Melaku S, Getahun A, Mengestou S, et al (2022) Potential live feeds for larval fish culture in Ethiopia. African J. Aquat. Sci. 47:423–435
Mohammad Mirzaie MA, Kalbasi M, Mousavi SM, Ghobadian B (2016) Investigation of mixotrophic, heterotrophic, and autotrophic growth of Chlorella vulgaris under agricultural waste medium. Prep Biochem Biotechnol 46:150–156
Munro PD, Henderson RJ, Barbour A, Birkbeck TH (1999) Partial decontamination of rotifers with ultraviolet radiation: The effect of changes in the bacterial load and flora of rotifers on mortalities in start-feeding larval turbot. Aquaculture 170:229–244
Nagappan S, Das P, AbdulQuadir M, et al (2021) Potential of microalgae as a sustainable feed ingredient for aquaculture. J Biotechnol 341:1–20
Ogello EO, Hagiwara A (2015) Effects of Chicken Manure Extract on the Population Growth, Mixis Induction and Body Size of the Freshwater Rotifer Brachionus angularis Gosse 1851. Asian Fish Sci 28:174–185
Ogello EO, Wullur S, Hagiwara A (2019) Blending fishwastes and chicken manure extract as low-cost and stable diet for mass culture of freshwater zooplankton, optimized for aquaculture. IOP Conf Ser Mater Sci Eng 567:12–22
øie G, Reitan KI, Olsen Y (1994) Comparison of rotifer culture quality with yeast plus oil and algal-based cultivation diets. J Eur Aquac Soc 2:225–238
Pszczółkowska A, Pszczółkowski W, Romanowska-Duda Z (2019) Potential of Chlorella vulgaris culture for waste treatment from anaerobic biomass biodigestion at the Piaszczyna (Poland) integrated facility. J Phycol 55:816–829
Rajagopal R, Mousavi SE, Goyette B, Adhikary S (2021) Coupling of microalgae cultivation with anaerobic digestion of poultry wastes: Toward sustainable value added bioproducts. Bioengineering 8:1–13
Richmond A, Hu Q (2013) Handbook of Microalgal Culture: Applied Phycology and Biotechnology: Second Edition. Blackwell Publishing Ltd.
Rico-martinez R, Dodsonb SI (1992) Culture of the rotifer Brachionus calyciflorus Pallas. Aquaculture 105:191–199
Sagar K, Sanjay KJ, Parmod KJ (2019) Nutrient content in compost from water hyacinth and its effect on germination and growth of wheat. J Handayama Geogr Archaeol 7:151–158
Samani ZA (2007) Producing liquid organic fertilizer from organic substrates. 5:1229–1238
Seaman MT, Gophen M, Cavari BZ, Azoulay B (1986) Brachionus calyciflorus Pallas as agent for the removal of E. coli in sewage ponds. Hydrobiologia 135:55–60
Skjermo J, Vadstein O (1993) Characterization of the bacterial flora of mass cultivated Brachionus plicatilis. Hydrobiologia 255–256:185–191
Starkweather PL, Gilbert JJ, Frost TM (1979) Bacterial feeding by the rotifer Brachionus calyciflorus: Clearance and ingestion rates, behavior and population dynamics. Oecologia 44:26–30
Suantika G, Dhert P, Rombaut G, et al (2001) The use of ozone in a high density recirculation system for rotifers. Aquaculture 201:35–49
Takaoka O, Ji SC, Ishimaru K, et al (2011) Effect of rotifer enrichment with herbal extracts on growth and resistance of red sea bream, Pagrus major (Temminck & Schlegel) larvae against Vibrio anguillarum. Aquac Res 42:1824–1829
Tan XB, Lam MK, Uemura Y, et al (2018) Semi-continuous cultivation of Chlorella vulgaris using chicken compost as nutrients source: Growth optimization study and fatty acid composition analysis. Energy Convers Manag 164:363–373
Tanasomwang V, Muroga K (1992) Effect of sodium nifurstyrenate on the reduction of bacterial contamination of rotifers (Brachionus plicatilis). Aquaculture 103:221–228
Verdonck L, Grisez L, Sweetman E, et al (1997) Vibrios associated with routine productions of Brachionus plicatilis. Aquaculture 149:203–214
Wang L, Chen L, Wu S (Xiao) (2020) Microalgae Cultivation Using Screened Liquid Dairy Manure Applying Different Folds of Dilution: Nutrient Reduction Analysis with Emphasis on Phosphorus Removal. Appl Biochem Biotechnol 192:381–391
Wang L, Li Y, Chen P, et al (2010) Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresour Technol 101:2623–2628
Watanabe WO, Alam S, Ostrowski AD, et al (2016) Live prey enrichment and artificial microdiets for larviculture of Atlantic red porgy Pagrus pagrus. Aquac Reports 3:93–107
Yan DC, Dong SL, Huang J, Zhang JS (2007) White spot syndrome virus (WSSV) transmission from rotifer inoculum to crayfish. J Invertebr Pathol 94:144–148
Yirga E, Brook L (2018) Zooplankton communities as an indicator of ecosystem productivity in Lake Tinishu Abaya, Rift Valley, Ethiopia. Int J Fish Aquac 10:53–70
Yu JP, Hino A, Noguchi T, Wakabayashi H (1990) Toxicity of Vibrio alginolyticus on the Survival of the Rotifer Brachionus plicatilis. Nippon Suisan Gakkaishi 56:1455–1460
Zhou 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
Zhu T, Jia H, Zhang H, et al (2024) Effects of chicken manure extract on the directed cultivation of bio-bait Chlorella vulgaris-rotifer (Brachionus plicatilis) and their fatty acid content. Aquaculture 581:740387

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

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Experimental set up

2.2 Microalgae strain and culture conditions

2.3 Organic media processing and preparation

2.4 Rotifer strain and culture conditions

2.4 Total bacterial counts

2.4 Water quality parameters

2.5 Data analysis

3. Results

3.1 Effects of partial substitution of BBM with organic media on the growth of Chlorella sp.

3.2 Effects of Chlorella sp. cultured on different organic media on the population growth of B.calyciflorus

3.3 Microbial loads of B.calyciflorus fed on Chlorella sp. cultured with organic media substitution

3.4 Water quality parameters

4. Discussion

4.1 Chlorella sp. culture performance on different organic media substitutions

4.3 Bacterial load of B.calyciflorus reared on Chlorella sp. cultured on different organic media

5. Conclusion and recommendation

Declarations

Competing interests:

Funding:

Author Contribution

Acknowledgement

Data availability:

References

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