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Rapid Production Biofloc by Inoculating Chlorella pyrenoidosa in a Separate Way

Zhejiang Provincial Key Laboratory of Aquatic Resources Conservation and Development, School of Life Science, Huzhou University, Huzhou 313002, China
Authors to whom correspondence should be addressed.
Water 2023, 15(3), 536;
Submission received: 31 December 2022 / Revised: 21 January 2023 / Accepted: 25 January 2023 / Published: 29 January 2023
(This article belongs to the Special Issue Water Quality in Aquaculture Production)


Microalgae play an important role in the formation of biofloc. To demonstrate the feasibility of Chlorella pyrenoidosa in biofloc formation, an experiment was performed with a simple random design consisting of five inoculation levels (in triplicate) of C. pyrenoidosa (0, 1 × 108, 1 × 109, 5 × 109, and 1 × 1010 cells·L−1) in the biofloc system. All treatments kept a C:N ratio of approximately 15:1. This study observed the effects of different initial concentrations of C. pyrenoidosa on biofloc formation, water quality and bacterial community in biofloc systems. The results indicated that C. pyrenoidosa had the ability to enhance biofloc development, especially when the C. pyrenoidosa initial concentration reached 5~10 × 109 cells·L−1. Too high or too low a concentration of C. pyrenoidosa will adversely affect the formation of biofloc. The effect of C. pyrenoidosa addition on water quality (TAN, NO2-N, and NO3-N) was not significant in the final stage. The inoculation of C. pyrenoidosa decreased the species richness and diversity of the bacterial community but increased the domination of Proteobacteria and Bacteroidota in the biofloc system, especially the order of Rhizobiales. The addition of C. pyrenoidosa could maintain water quality by increasing the proportion of several denitrifying bacteria, including Flavobacterium, Chryseobacterium, Pseudomonas, Brevundimonas, Xanthobacter, etc. These above dominant denitrifying bacteria in the biofloc system could play a major role in reducing the concentration of NO2-N and NO3-N. So, we recommended the reasonable concentration is 5~10 × 109 cells·L−1 if C. pyrenoidosa is used to rapidly produce biofloc.

1. Introduction

The aquaculture industry plays an important role in providing aquatic products for the world population through intensive cultivation. However, as aquaculture cultivation intensifies, researchers found that only 20–30% of nitrogen is utilized and retained in organisms [1]. That is, around 70–80% of nitrogen is released into the adjacent environment, which could produce an adverse effect [2,3]. Biofloc technology (BFT) was considered to play an important role in maintaining the rearing water quality and providing extra nutrition for aquaculture animals [4,5]. This technology could solve the adverse effect from nitrogen discharge by constructing biofloc systems [6,7]. This system is generally constructed by providing organic carbon and maintaining the C/N ratio at a reasonable level to remove toxic nitrogen [8,9]. As a microbial-based system, its structure is considered to contain 29% microalgae, 35% bacteria, 24% fungi and 12% zooplankton [10]. Microalgae, as one of the main components, is involved in the formation of biofloc. For instance, Chlorophyceae, Bacillariophyceae, and Cyanophyceae were discovered as the most common species in the initial stages of the biofloc system [11,12]. In addition, Chlorella, Acutodesmus and Chlamydomonas have been regularly found to be a stable biofloc composition [13]. These microalgae not only participate in the formation but also play a key role in removing nitrates, providing oxygen to heterotrophic bacteria and enhancing the aggregation of microorganisms in the water by their excreta [14,15,16]. Many studies thought that the enhancing effect of microalgae is important in the formation of biofloc. Loria et al. [17] found that the addition of Chlorella vulgaris, Chlorella sorokiniana, Scenedesmus dimorphus, and Neochloris oleoabundans could form biofloc. The addition of Chlorella sp., Grammatophora sp., and Navicula sp. could increase the quantity of protein and lipids in biofloc [17,18]. It was proven that the improvement of C. vulgaris on floc was caused by secreting extracellular metabolites, which could provide a high level of flocculating activities [19,20]. This suggested that the choice of appropriate species could be a key factor that influenced the effect of biofloc formation. However, these studies were conducted using conventional production, culturing biofloc in the same units with farmed animals [21]. To focus on the quality of the biofloc and facilitate the quantification of biofloc production, the biofloc needs to be cultured in a separate way [22,23].
Compared with C. vulgaris, Chlorella pyrenoidosa (C. pyrenoidosa) was found to produce a higher biomass [24]. It is a widely distributed and cultivated freshwater microalga that is extensively used in aquaculture as food for aquaculture animals [25]. According to the Food and Agriculture Organization (FAO), C. pyrenoidosa is one of the healthy green foods, rich in nutrients such as protein, total lipids and total carbohydrates [26]. In addition, C. pyrenoidosa is characterized by a high reproduction rate and the ability to use carbon sources for mixotrophic growth [27,28], especially the strong ability to convert ammonia to high nutritional biomass under a mixotrophic mode [29]. This evidence indicated that C. pyrenoidosa was regarded as the most potentially promising species for producing biofloc. However, the effects of C. pyrenoidosa on the formation of biofloc are still unknown. So, we explored the effects of different initial concentrations of C. pyrenoidosa on the formation amount, water quality and bacterial community under the novel separate production of biofloc. The aim of this study was to obtain which initial concentration of C. pyrenoidosa would be suitable for the formation of biofloc. We tried to reveal the promotion mechanism of C. pyrenoidosa on biofloc through water quality and bacterial community. This is vital in providing some fundamentals for elaborating the rapid establishment of biofloc by inoculating C. pyrenoidosa.

2. Materials and Methods

2.1. Microalgae and Bacteria Strain

C. pyrenoidosa (FACHB-9) was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). This microalga was grown in BG11 medium sterilized at 121 °C for 20 min. The cultivation was performed at 25 ± 1 °C with a 12:12 light-dark cycle. In the study of biofloc formation, Bacillus subtilis, as a kind of probiotic bacterium in aquaculture, was widely applied to improve the formation process [30,31]. So, we selected this bacterium as an inoculating strain for our biofloc system under separate production. B. subtilis was obtained from Baiwo co., Ltd. (Beijing, China) and was activated by inoculating it into ultra-water under sterile conditions and culturing it at 37 °C for 24 h with agitation.

2.2. Experiments Design

The experimental system consisted of a reactor (measuring cup, 3 L), a cylindrical aeration stone and an air pump. Five treatments were set up for the current experiments, including different inoculation concentrations of C. pyrenoidosa (0, 1 × 108, 1 × 109, 5 × 109 and 1 × 1010 cells·mL−1) with three replications (Table 1). B. subtilis was added to each reactor to a final concentration of 2 × 107 CFU·mL−1, which was adjusted on the basis of our pre-experiments based on the results of Yusufi et al. [32]. All treatments were run at 25 ± 1 °C and in a 12:12 light-dark cycle for 13 days. Glucose was used in all treatments as a carbon source, while urea was used as a nitrogen source. The ratio of carbon and nitrogen (15:1) was maintained through the additional carbon and nitrogen source. No water exchange was carried out in the biofloc treatments; however, the regular addition of freshwater was performed to replace water loss due to evaporation. During a 13-day experimental period, 100 mL of water in biofloc reactors was collected and filtered through a 0.45 μm pore size membrane under vacuum pressure through a pump. Then, filter papers were used to determine the total suspended solid (TSS) [33], and the filtrate was used to analyze the water quality.

2.3. Analytical Methods

2.3.1. Biofloc Development Analysis

Changes in TSS concentrations and turbidity over time can indicate the biofloc development in water [34]. To determine TSS, an unused filter of 0.45 μm pore size was dried in a dryer at 105 °C and then weighed on an electronic microbalance. The filter was dried after filtering a 100 mL biofloc water sample. The net dry weight was calculated by subtracting the weight of the unused filter from the final weight [35]. The turbidity was tested with the turbidity meter (430IR, Turb, MUC, GER).

2.3.2. Water Quality Analysis

Water quality parameters, including total ammonia nitrogen (TAN), nitrite nitrogen (NO2-N), nitrate nitrogen (NO3-N), temperature (T), pH and dissolved oxygen (DO), were measured once every two days. The concentration of TAN, NO2-N, and NO3-N were analyzed using a flow-injection analyzer (QC8500, Hach, Loveland, CO, USA). The physicochemical variables of T (°C), pH, and DO (mg·L−1) were measured in situ using the multiparameter (ProQuatro, YSI, Yellow Springs, OH, USA).

2.3.3. Bacterial Community Analysis

To investigate the impact of C. pyrenoidosa on bacterial community composition more comprehensively, three time points were chosen to analyze the bacterial community. Water samples were filtered through a 0.22 μm pore size membrane and stored frozen at −80 °C for further analysis. These samples were sent to Yuanxin Co. (Shanghai, China) for the sequencing of the 16S rDNA V4-V5 region and biofloc bacterial diversity analysis (primer sequence information, 515F: 5′-GTGCCAGCMGCCGCGG-3′ and 907R: 5′-CGGTCAATTCMTTTRAGTTT-3′) [5]. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (version 7.0.1090). The bacterial diversity was measured using Shannon and Simpson indexes, while the Chao1 and Ace indexes were used to reflect species richness. These four parameters were determined based on the calculated OTUs and calculated in Qiime (Version 1.9.0).

2.4. Statistical Analysis

Data were shown as the average value ± standard deviation. SPSS (Version 25.0) was used to process the data. One-way analysis of variance (ANOVA) was used to examine significant differences among the treatments of samples. Differences were considered significant when p < 0.05. The Duncan test was adopted for post hoc multiple comparisons if there was a significant difference among data. CCA (canonical correspondence analysis) was performed with the top 10 dominants at the genus level to clarify the relationship between environmental factor dynamics and bacterial community variations.

3. Results

3.1. Biofloc Formation Differences under Different C. pyrenoidosa Concentration

The biofloc development over time is shown in Figure 1. Both TSS (Figure 1a) and turbidity (Figure 1b) levels increased gradually throughout the experiment period. There were significant differences between the experimental treatments and the control treatment. The TSS and turbidity no longer changed significantly after the 11th day (p > 0.05). At the end of the experiments, the maximum TSS and turbidity were observed in treatment C, reaching about 535.02 ± 95.52 mg·L−1 and 110.03 ± 13.80 NTU, respectively. The least TSS and turbidity were reported with the control treatment, recording 293.00 ± 50.91 mg L−1 and 84.67 ± 11.82 NTU, respectively. Following these results, C. pyrenoidosa had the ability to enhance biofloc development. The promotional effect was the most obvious when the C. pyrenoidosa initial concentration reached 5~10 × 109 cells·L−1.

3.2. Change in Water Quality under Different C. pyrenoidosa Concentration

The water quality parameters of experimental and control treatment are shown in Table 2. The water temperature, pH and DO in all treatments remained stable and were within the ideal range throughout the trial.
The concentration of TAN, NO2-N and NO3-N in five treatments during the 13-day experimental period are given in Figure 2. The TAN (Figure 2a) was not detected on day 1. The TAN was gradually increased and then maintained a relatively stable state throughout the experiment. There was no significant difference in the TAN concentration among treatments in the late stage of the culture (p < 0.05). The NO2-N (Figure 2b), showing no significant difference (p < 0.05) among treatments in the initial and late stages, fluctuated within a relatively low level during the middle stage. High values for NO3-N were seen at the start of the culture (Figure 2c). The NO3-N was gradually decreased and then became stagnant throughout the 13-day trial in treatments A, B, C and E. In contrast, treatment D showed accumulation during the experiment.

3.3. Dynamic Structure of the Bacterial Community under Different C. pyrenoidosa Concentration

The species richness and diversity of the bacterial community are shown in Table 3. The Chao1 and Ace indexes gradually increased over time in all treatments, while the control treatment had a higher species richness than the experimental treatments. The Simpson and Shannon indices in the experimental treatments were lower than the control treatment at the same sampling time. In summary, the inoculation of C. pyrenoidosa decreased the species richness and diversity of bacterial community in the biofloc system.
The results of the bacterial community structure are given in Figure 3. At the phylum level (Figure 3a), A, B, C, D and E mostly consisted of Proteobacteria (62%, 70%, 55%, 54% and 52%), Bacteroidota (26%, 22%, 25%, 29% and 24%) and Firmicutes (5%, 1%, 2%, 1% and 6%). At the order level (Figure 3b), A, B, C, D and E mainly consisted of Flavobacteriales (17%, 14%, 20%, 20% and 14%), Rhizobiales (5%, 5%, 17%, 25% and 7%), Caulobacterales (11%, 27%, 8% 5% and 8%), Pseudomonadales (10%, 6%, 4%, 3% and 13%) and Sphingomonadales (6%, 8%, 7%, 7% and 1%). The predominant genus is shown in Figure 3c. There were many unidentified species which illustrated the complexity of bacterial communities in biofloc systems. A, B, C, D and E mostly consisted of Flavobacterium (14%, 9%, 17%, 20% and 15%), Brevundimonas (11%, 32%, 9%, 5% and 2%), Chryseobacterium (8%, 9%, 12%, 11% and 4%), Pseudomonas (11%, 6%, 5%, 4% and 5%) and Xanthobacter (3%, 3%, 10%, 10% and 7%).
The result of CCA is shown in Figure 4. A positive correlation existed between TSS, turbidity and TAN, whereas a negative correlation existed between these variables and NO2-N and NO3-N. Xanthobacter, Flavobacterium and Brevundimonas were influenced by TSS and turbidity. NO2-N had a positive relationship with Fimbriimonadaceae-norank. The dominant bacteria that were positively associated with NO3-N included Sphingomonas, Chryseobacterium, Pseudomonas and Enterobacteriaceae-unclassified. Elstera and Legionella were influenced by TAN.

4. Discussion

4.1. Influence of C. pyrenoidosa on Biofloc Development

When the concentration of phytoplankton in the biofloc system increases, the amount of biofloc formation increases [36]. This statement is confirmed by the results that show the Chlorella sp. inoculum enhances the stability of biofloc formation and makes biofloc production higher [19]. So, our results indicated that C. pyrenoidosa could promote biofloc formation. It is very reasonable to show similar characteristics for C. pyrenoidosa, which is considered one of the important microalgae of Chlorella sp. From a macro perspective, we also found that it had no significant effect on the biofloc formation when the initial addition of C. pyrenoidosa was below 1 × 108 cells·L−1. This finding is consistent with the result of previous research [37], which might be attributed to the lack of influence of the lower concentration of Chlorella sp. (<1 × 109 cells·L−1) on TSS concentration [38]. However, this microalga still can affect the particle size of biofloc from a microscopic perspective [38]. When the initial addition of C. pyrenoidosa exceeded 1 × 108 cells·L−1, it could significantly promote the biofloc formation. Nevertheless, the biofloc system with C. pyrenoidosa addition (1 × 1010 cells·L−1) demonstrated lower production during the final phase. This may have been due to the fact that a high TSS concentration reduces the light entrances into the system, intervenes in the growth of C. pyrenoidosa [39] and inhibits the growth of other phytoplankton [40].

4.2. Influence of C. pyrenoidosa on Water Quality

In the present study, the addition of C. pyrenoidosa did not affect TAN, NO2-N and NO3-N. The same evidence was revealed in the Chlorella sp. addition, which did not significantly influence TAN and NO2-N [41]. Although Chlorella sp. generally has the ability to utilize carbon sources for growth to remove or uptake TAN [42], it does not have enough ability to transform TAN through the production of bacterial biomass for a large amount of glucose added [43]. Thus, it could have been caused by homologous reasons as C. pyrenoidosa is one of the dominant microalgae of Chlorella sp. Additionally, TAN showed an increasing trend until the end of all treatments, which was mainly due to the degradation of dead biomass [23] and the development of denitrifying bacteria during biofloc formation. Contrary to TAN changes, NO3-N went down in four treatments except treatment D, which could have happened due to the following three mechanisms: aerobic heterotrophic denitrification into nitrogen gas [19], dissimilatory nitrate reduction to ammonium (DNRA) [44] and heterotrophic assimilated into bacterial biomass [45].

4.3. Influence of C. pyrenoidosa on Bacterial Community

The observation that microalgae reduced bacterial richness and diversity in biofloc systems has also been reported [46]. We further formalized the above view in our research about the addition of C. pyrenoidosa. However, the dominant bacteria were indeed selective in the biofloc formation. In this condition of high carbon, Proteobacteria could produce a variety of metabolic species that could degrade organic matter as well as remove nutrients [47]. We have found that Pseudomonadales, Caulobacterales and Sphingomonadales belonging to the phylum of Proteobacteria were the four main orders playing a part in the above metabolic process. In particular, the order of Rhizobiales was proved to be conducive to the nutrient cycling and organic compounds’ utilization in biofloc systems [48,49]. In addition, the order of Flavobacteriales belonging to the phylum of Bacteroidota also has a high capacity to use organic compounds [50]. A similar phenomenon has been verified in other studies [5,51]. All of this could reasonably explain why the two bacteria (Proteobacteria and Bacteroidota) at the phylum level were dominant in our research and why the relative abundance of Rhizobiales at the order level increased with the increasing concentration of C. pyrenoidosa. Meanwhile, several denitrifying bacteria were considered as the main bacteria in the heterotrophic and aerobic conditions designed in this study. Under this circumstance, as the members of Bacteroidota, Flavobacteriales (Flavobacterium) were not only associated with heterotrophic denitrification [52] and the accumulation of nitrogen [53] but also had the ability to increase the flocculation efficiency of several green algae cultures [54]. In addition, Chryseobacterium was considered to be capable of utilizing NO3-N aerobically in the presence of NH4+-N [55]. We also found that Pseudomonas, Brevundimonas and Xanthobacter were the dominant genera in the phylum of Proteobacteria. Pseudomonadales (Pseudomonas) had the characteristics of heterotrophic nitrification and aerobic denitrification simultaneously [56], while Brevundimonas could perform aerobic denitrification with high nitrate [57,58]. Xanthobacter also have the ability to make the dissimilatory reduction of both NO3-N and NO2-N to gaseous forms of nitrogen [59]. This evidence could also reasonably explain why these above bacteria at the genus level were dominant in our research.

5. Conclusions

The present research clearly demonstrated that the addition of C. pyrenoidosa could promote the production efficiency of the biofloc. Especially when the initial concentrations of C. pyrenoidosa were in a reasonable range of 5~10 × 109 cells·L−1, it was the better-inoculated level for the rapid formation and stability of the biofloc. Too low a concentration could not produce a macroscopically visible effect, while too high a concentration would inhibit the performance of the biofloc production. It is an important point that needs to be taken into account for the higher TSS concentration, which could reduce the light entrances into the system and intervene in the photosynthesis and the growth of microalgae. We also proved that the addition of C. pyrenoidosa could bring about the reduction in species richness and diversity. At the same time, the inoculation of this microalgae could make Proteobacteria and Bacteroidota dominant in the biofloc system, while the order of Rhizobiales could be a main biological factor promoting biofloc formation. In the process of biofloc formation, the concentration of TAN over time was raised under the addition of C. pyrenoidosa, which could be ascribed to the existence of three functions: the transformation of TAN, the degradation of dead biomass, and denitrification in the biofloc system. CCA analysis proved that the amount of biofloc formation was negative with NO2-N and NO3-N, as several dominant denitrifying bacteria in the biofloc system played a major role in the formation. Furthermore, the inoculation of C. pyrenoidosa could maintain water quality by increasing the proportion of several denitrifying bacteria (such as Flavobacterium, Chryseobacterium, Pseudomonas, Brevundimonas, Xanthobacter, etc.).

Author Contributions

All authors listed have contributed to this study. Conceptualization, R.Z. and J.Z.; methodology, Y.C., Z.F. and Z.S.; software, Y.C. and R.Z.; validation, R.Z., J.Z., Q.X. and Y.Z.; formal analysis, J.Z., Q.X. and Y.Z.; investigation, R.Z.; resources, R.Z. and Y.Z.; data curation, Z.F. and Z.S.; writing—original draft preparation, Y.C.; writing—review and editing, Y.C., R.Z. and Y.Z. All authors have read and agreed to the published version of the manuscript.


The research was financially supported by the Public Welfare Research Key Project of Huzhou City (2019GZ28), the Basic Public Welfare Research Project of Zhejiang Province (LQ21C190001), the Natural Science Foundation of Zhejiang Province (LTGN23C190002) and the Innovation and Entrepreneurship Training Project for College Students (202001170).

Data Availability Statement

The data that support the findings of this research are available from the corresponding author (Zhang, R.), upon reasonable request.


Thanks to Zhejiang Provincial Key Laboratory of Aquatic Resources Conservation and Development for their assistance and infrastructure, and Yilin He and Zhongtao Deng for their support for sampling activity along this experiment. This paper has been professionally checked and corrected by Ting Wang.

Conflicts of Interest

The authors declare no conflict of interest.


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Figure 1. The variation in (a) total suspended solids (TSS) levels and (b) turbidity of five treatments.
Figure 1. The variation in (a) total suspended solids (TSS) levels and (b) turbidity of five treatments.
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Figure 2. The variation in (a) total ammonia nitrogen (TAN), (b) nitrite nitrogen (NO2-N) and (c) nitrate nitrogen (NO3-N) of five treatments.
Figure 2. The variation in (a) total ammonia nitrogen (TAN), (b) nitrite nitrogen (NO2-N) and (c) nitrate nitrogen (NO3-N) of five treatments.
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Figure 3. Bacterial community at (a) phylum, (b) order and (c) genus levels of five treatments.
Figure 3. Bacterial community at (a) phylum, (b) order and (c) genus levels of five treatments.
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Figure 4. Canonical correspondence analysis (CCA) with water parameters and bacterial community (top 10 dominant at genus level) in biofloc system. TAN, total ammonia nitrogen; NO2-N, nitrite nitrogen; NO3-N, nitrate nitrogen; TSS, total suspended solid; Turb, turbidity. The water parameters were indicated as arrows. The dominant bacteria were represented as lozenges. Samples were indicated as circles, with numbers corresponding to their sampling time.
Figure 4. Canonical correspondence analysis (CCA) with water parameters and bacterial community (top 10 dominant at genus level) in biofloc system. TAN, total ammonia nitrogen; NO2-N, nitrite nitrogen; NO3-N, nitrate nitrogen; TSS, total suspended solid; Turb, turbidity. The water parameters were indicated as arrows. The dominant bacteria were represented as lozenges. Samples were indicated as circles, with numbers corresponding to their sampling time.
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Table 1. C. pyrenoidosa initial inoculation concentrations of the treatments in experiment.
Table 1. C. pyrenoidosa initial inoculation concentrations of the treatments in experiment.
C. pyrenoidosa (cells·L−1)1 × 1081 × 1095 × 1091 × 10100
B. subtilis (CFU·mL−1)2 × 1072 × 1072 × 1072 × 1072 × 107
Volume (L)33333
Table 2. Water quality parameters in five treatments throughout the 13-day experimental period.
Table 2. Water quality parameters in five treatments throughout the 13-day experimental period.
TreatmentspHDO (mg·L−1)Temperature (°C)
A8.38 ± 0.355.84 ± 1.3725.19 ± 0.32
B8.55 ± 0.306.46 ± 0.9425.28 ± 0.24
C8.46 ± 0.426.77 ± 1.1425.34 ± 0.20
D8.56 ± 0.376.25 ± 0.7825.32 ± 0.39
E8.37 ± 0.345.55 ± 1.1324.32 ± 0.49
Note: Each value represents mean ± SD.
Table 3. The richness and diversity of the bacterial community from all treatments at different sampling times.
Table 3. The richness and diversity of the bacterial community from all treatments at different sampling times.
Chao 1AceShannonSimpson
1dA1006.67 ± 52.541405.67 ± 69.371871.00 ± 87.682.89 ± 0.170.12 ± 0.01
B1421.00 ± 48.081988.50 ± 86.972084.00 ± 43.843.18 ± 0.290.12 ± 0.01
C1272.00 ± 59.401662.67 ± 118.371905.00 ± 37.643.35 ± 0.150.09 ± 0.01
D1019.50 ± 96.871796.00 ± 100.411922.00 ± 179.613.48 ± 0.200.08 ± 0.01
E2556.00 ± 115.973651.00 ± 224.863531.67 ± 364.533.64 ± 0.180.10 ± 0.01
7dA990.00 ± 123.041592.00 ± 96.171697.50 ± 164.763.55 ± 0.040.09 ± 0.01
B1441.67 ± 94.732007.67 ± 109.042158.67 ± 130.603.33 ± 0.310.17 ± 0.01
C1350.67 ± 36.461947.33 ± 79.122508.67 ± 76.873.64 ± 0.240.06 ± 0.01
D1297.50 ± 36.062035.00 ± 104.652745.67 ± 242.843.63 ± 0.240.08 ± 0.00
E3290.67 ± 240.784200.33 ± 107.304350.00 ± 120.394.77 ± 0.190.06 ± 0.00
13dA1543.00 ± 183.852040.50 ± 293.452207.50 ± 300.523.23 ± 0.310.19 ± 0.01
B1466.33 ± 77.911956.33 ± 87.812143.67 ± 90.183.30 ± 0.290.16 ± 0.01
C1716.00 ± 50.912336.67 ± 203.012504.33 ± 208.714.10 ± 0.210.07 ± 0.01
D2385.00 ± 168.293114.00 ± 290.563759.67 ± 370.093.85 ± 0.210.10 ± 0.00
E3626.00 ± 113.374568.67 ± 37.454700.33 ± 66.915.32 ± 0.310.04 ± 0.01
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Chen, Y.; Fu, Z.; Shen, Z.; Zhang, R.; Zhao, J.; Zhang, Y.; Xu, Q. Rapid Production Biofloc by Inoculating Chlorella pyrenoidosa in a Separate Way. Water 2023, 15, 536.

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Chen Y, Fu Z, Shen Z, Zhang R, Zhao J, Zhang Y, Xu Q. Rapid Production Biofloc by Inoculating Chlorella pyrenoidosa in a Separate Way. Water. 2023; 15(3):536.

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Chen, Yang, Zhichao Fu, Zhenyi Shen, Rongfei Zhang, Jianhua Zhao, Yixiang Zhang, and Qiyou Xu. 2023. "Rapid Production Biofloc by Inoculating Chlorella pyrenoidosa in a Separate Way" Water 15, no. 3: 536.

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