Effect of recycling the culture medium on biodiversity and population dynamics of bio-contaminants in Spirulina platensis mass culture systems

Algal Research 44 (2019) 101718

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Effect of recycling the culture medium on biodiversity and population dynamics of bio-contaminants in Spirulina platensis mass culture systems Danni Yuana,b, Mimi Yaoa,b, Lan Wanga,b, Yanhua Lia,b, Yingchun Gonga,b, , Qiang Hua,b,c,d,e, ⁎

T ⁎

a

Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Key Laboratory for Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China c SDIC Microalgae Biotechnology Center, SDIC Biotech Investment Co., LTD., Beijing 065200, China d Beijing Key Laboratory of Algae Biomass, Beijing 100142, China e Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China b

ARTICLE INFO

ABSTRACT

Keywords: Spirulina cultivation Microzooplankton Amplicon sequencing Recycled medium

Spirulina (Arthrospira) is an important microalga that can generate a variety of commercial products. A semicontinuous mode of cultivation is often employed in microalgal cultivation. The traditional view, however, has been that cultures with recycled medium are more likely to be contaminated by microzooplankton, which is regarded as one of the critical problems in mass algal cultivation. In this study, the relationship between the population dynamics of contaminants and the medium conditions was investigated in Spirulina cultures. Spirulina platensis was cultivated in three groups of raceway ponds: one with fresh culture medium, one with medium that had been recycled for a month, and the third with medium recycled for six months. The results showed that totally 13 species of microzooplankton were observed by light microscopy, and 42 operational taxonomic units (OTUs) of prokaryotic contaminants were detected using amplicon sequencing. Out of all the contaminants, Brachionus plicatilis and Euplaesiobystra hypersalinica were observed to be the most harmful species in Spirulina cultures, while Proteobacteria were the most commonly found non-Cyanobacteria OTUs. On the initial day, more species of microzooplankton were introduced to the cultures that had recycled medium (9 species) than to those that had fresh medium (5 species). By the end of the cultivation, the algal biomass in the fresh medium group was the highest (2.8 g L−1), being almost 5 times higher than in the other two groups (around 0.50 g L−1). Our results proved that Spirulina can grow the best with fresh medium and that the more the culture medium is recycled, the stronger the inhibition on the growth of microalgae and microzooplankton. In order to improve large-scale Spirulina production, it is necessary to both subject the recycled medium to appropriate treatment to reduce the presence of harmful predators and to find effective ways to control contaminants.

1. Introduction

use of nutrients and water, and hence to reduce the cost [9]. Ho et al. [10] report that compared to batch culture, semi-continuous cultivation can reduce the limitations to growth related to nutrients and light penetration during the later stages of cultivation. Furthermore, Yang et al. [11] analyzed one batch of biodiesel production from microalgae and showed that the recycling of water from harvested culture reduces the water and nutrient usage by 84% and 55%, respectively. On the other hand, long-term semi-continuous cultivation can also result in economic losses. The use of recycled medium frequently leads to a loss of productivity, possibly due to the accumulation of secondary metabolites, non-consumed nutrients and cellular debris in the cultures [12]. More importantly, the use of the recycled medium introduces the risk of biological contamination. Semi-continuous cultivation makes the microalgal cultures more susceptible to contamination with predator

The edible cyanobacterium Spirulina (Arthrospira) has attracted attention due to its commercial production and application as food, as aquaculture feed, in cosmetics and for nutraceutical purposes [1–3]. Spirulina was first found in Mexico in the 16th century, but after that little attention was paid to it until the 1960s [4]. Spirulina cultivation companies started to develop in China in the early 1990s. By the mid1990s, China was producing more Spirulina than any other country in the world [5,6], producing over 1.0 × 104 tons of Spirulina biomass per annum, which represents > 70% of the global supply [7]. For environmental and economic reasons, semi-continuous cultivation is the mode widely applied for Spirulina outdoor cultivation [8]. Semi-continuous cultivation is employed in an attempt to reduce the

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Corresponding authors at: Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. E-mail addresses: [email protected] (Y. Gong), [email protected] (Q. Hu).

https://doi.org/10.1016/j.algal.2019.101718 Received 3 June 2019; Received in revised form 10 October 2019; Accepted 1 November 2019 Available online 15 November 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.

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microzooplankton, which is regarded as one of the critical problems in the commercial large-scale production of microalgae [9]. A diverse assemblage of fungi, bacteria, viruses, ciliates, amoebae, rotifers and other zooplankton are known to attack microalgal cultures [13–15]. For example, Chlorella sp. has often been found to be severely constrained by repeated contamination with and grazing by Poterioochromonas malhamensis [16]. Additionally, Ganuza et al. [17] have observed that Chlorella cultures can be seriously contaminated with the bacterium Vampirovibrio chlorellavorous. For Scenedesmus, the culture quality and productivity declines severely when it is invaded by the chytrid Rhizophidium sp. [18], the amoeba Vernalophrys algivore [19] or the ciliate Loxodes magnus [20]. For Spirulina cultivation, it has usually been thought that not many bio-contaminants could grow in the microalgal cultures, because of their high alkalinity and high pH value. However, we surveyed eight Spirulina production companies throughout China and found that all the cultures were contaminated with and grazed by the rotifer Brachionous plicatilis, which was considered as the most harmful species for Spirulina cultivation, and a total of 23 species of microzooplankton were observed with microscopic observation [21,22]. Moreover, Shimamatsu [23] reported that some green algae and bacteria could contaminate Spirulina cultures from commercial Spirulina producers when cultivated under conditions of continuous recycling of the culture medium. Up until now, our understanding of biological contamination in Spirulina culture has been mainly based on morphological observations and sequencing of small subunit ribosomal RNA (SSU rRNA) genes for a small number of species [21]. Through investigation, we have learned that the probability of a decline in production or a complete collapse of culture due to contamination during the large-scale culture of Spirulina is much higher than that previously reported, and our current understanding about pollutants cannot fully explain the phenomenon of frequent collapse of Spirulina production. Now, however, amplicon sequencing can reveal the species compositions and relative abundance of prokaryotic microorganisms that cannot be revealed by traditional methods [24], and has been successfully applied to characterize the bacterial communities in Chlorella cultures [17], eukaryotic structures in Scenedesmaceae mass culture [18] and biological contaminants in the commercial production of Spirulina supplements [25]. Ecological studies of bio-contaminants in microalgal cultures are important for the development of risk management and remediation strategies for large-scale cultivation, with the ultimate goal being to ensure the sustainable high-yield production of microalgal biomass [26]. Microzooplankton are considered to be the most important and most common bio-contaminants, and their distribution is restricted by many environmental factors (e.g. salinity, nitrogen and phosphorus) which have been investigated comprehensively in natural aquatic ecosystems [27–29]. However, very few studies have been conducted on microzooplankton in mass algal cultures. Although we have found widespread contamination with microzooplankton in the Spirulina cultures of the eight companies in China [21], we lack a comprehensive understanding of the occurrence and development of microzooplankton in microalgal cultivation as a whole, and of the effect on Spirulina growth of different media recycling conditions. The purpose of the present study, therefore, was to characterize the bio-contaminants in Spirulina cultures under different media recycling conditions, and evaluate the resulting impact of the bio-contaminants on the algal biomass.

Fig. 1. The experimental site: location within China (main image) and outdoor culture pond with cover (inset, bottom right). Table 1 Environmental factors in Spirulina platensis cultures over six days of cultivation in raceway ponds under different media recycling conditions. Ponds

A

B

C

WT (°C) pH Cond. (μs cm−1) Salinity (‰) DO (mg L−1) PO4–P (mg L−1) NH4–N (mg L−1) NOX–N (mg L−1) DOC (mg L−1)

30.1 ± 0.1 9.50 ± 0.03 8.3 × 103 ± 56 4.14 ± 0.02 18.49 ± 0.54 52.0 ± 4.9 11.9 ± 5.2 82.3 ± 1.6 17.6 ± 7.8

29.0 ± 0.3 9.85 ± 0.03 8.6 × 103 ± 42 4.64 ± 0.21 11.39 ± 0.70 48.0 ± 1.4 8.37 ± 0.50 83.5 ± 2.2 46 ± 14

29.4 ± 0.2 9.53 ± 0.43 7.9 × 103 ± 29 4.0 ± 1.5 14.9 ± 4.7 43 ± 16 16.0 ± 5.4 73 ± 28 42 ± 13

*A, ponds with fresh medium (n = 2); B, ponds under semi-continuous cultivation with recycled medium that had been used for one month (n = 3); C, pond under semi-continuous cultivation with medium that had been used for six months (n = 2). Values are means of two or three biological replicates with standard deviations (SD). WT, water temperature; Cond., conductivity; DO, dissolved oxygen; NOXeN, the sum concentrations of nitrate and nitrite; DOC, dissolved organic carbon.

Zarrouk medium [30] in seven 7.0 × 102 m2 raceway ponds (1.0 × 102 m length, 7 m width, 0.23 m height) at 29 ± 1 °C in a greenhouse. The depth of the Spirulina culture ranged from 0.17 m to 0.27 m and the flow rate ranged from 0.17 cm s−1 to 0.46 cm s−1. Cultivation lasted for six days from 11th July (day 1) to 16th July (day 6) in 2014. The cultures were classified into three groups based on the medium used. Two ponds, classified as group A, were cultivated with fresh medium; for three ponds, classified as group B, semi-continuous cultivation was employed with recycled medium which had been used for a month; and for the final two ponds, classified as group C, the cultivation mode was semi-continuous with recycled medium which had been used for six months. In each case, a large initial inoculum of algae was obtained by filtering seed culture through a 1.0 × 103 μm net, to give an initial dry weight in each experimental culture of dry weights ranged from 0.20–0.40 g L−1. 2.2. Physicochemical analysis

2. Materials and methods

Temperature, salinity, pH, conductivity and dissolved oxygen (DO) were measured in situ by a multi-parameter probe (YSI Professional Plus, Yellow Springs, USA). Water depth and flow rate were measured by velocimeter (FP111, Global water, USA). Samples of culture (2.5 × 102 mL) for environmental analysis were filtered immediately after sampling through 0.45 μm Millipore filters. Concentrations of organic phosphorus (PO43−–P) and organic nitrogen (NH4+–N, NO3−–N,

2.1. Microalgal cultivation All the experimental cultivations were conducted at a commercial company producing Spirulina products in Dafeng city – a coastal city situated in the eastern Jiangsu province and northwest of Shanghai, China (Fig. 1). The microalga Spirulina platensis was cultivated using 2

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Fig. 2. Morphology of Spirulina platensis over six days of cultivation in raceway ponds. A and B, algae cultivated in fresh medium; C and D, algae cultivated in recycled medium (Scale bar=20 μm). Algae collected by 1000 μm filter. Table 2 Morphology of Spirulina platensis over six days of cultivation in raceway ponds. Group

Medium

Algaea

A B C

New medium Recycled medium for one month Recycled medium for six months

Moderate pitch; spiral number: 7–12; Flabby pitch; spiral number: 5–8; Flabby pitch (partly) & broken algal (partly); spiral number: 1–6;

A, B and C are as described in Table 1. a The cell length for all algae was 2.5–3.7 μm and cell width was 7.3–9.0 μm.

measurement of absorbance at 220 nm and at 275 nm, and NO2−–N was determined by the N-(1-naphthyl)-ethylenediamine dihydrochloride spectrophotometric method [31]. A TOC–L analyzer (Shimadzu Corporation, Japan) was used to determine dissolved organic carbon (DOC) concentration. 2.3. Measurement of algal growth The total dry weight (DW) of microalgal culture was measured according to Zhu [32]. Samples (25 mL) were collected each day and filtered through GF/C membrane (1.2 μm), with vacuum pressure differentials maintained at 35 to 55 mmHg. The filters were then dried at 105 °C to a constant weight, cooled down in a vacuum desiccator and weighed. 2.4. Identification and quantification of microzooplankton

Fig. 3. Growth of Spirulina platensis over six days of cultivation in raceway ponds, from 11th July to 16th July 2014. A, B and C, experimental groups of ponds are as described in Table 1. Data are shown as the mean of two or three biological replicates with error bars ( ± standard deviation).

2.4.1. Morphological observation Samples (15 mL) of algal culture were taken each day and examined by microscopy immediately after sampling. Observations and photomicrography of these live samples were performed with differential interference contrast under an Olympus BX53 microscope with a DP80 digital camera (Olympus, Japan). Rotifers were identified following Wang [33], while the identification of protozoa followed Tan [34], Shen [35] and Patterson [36]. Some contaminants had been isolated for SSU rRNA gene sequencing in our previous publication [21].

NO2−–N) were measured in the laboratory following standard protocols: PO43−–P was determined using the molybdate blue method, NH4+–N was determined colorimetrically with Nessler reagent, NO3−–N was determined according to a reference method based on 3

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Function groupa

Y

57

A, B

0.12

Agencourt AMPure beads (Beckman Coulter, USA) following the manufacturer's instructions. The amplicons were quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, USA) and then pooled. The Illumina adaptor ligation was performed using the NEBNext Quick Ligation Module (NEB, USA). Then, 16S rRNA tag-encoded highthroughput sequencing was carried out using an Illumina Miseq platform with the PE300 program at the Personal Biotechnology Company (Wuhan, China).

Table 3 The main contaminating microzooplankton (maximum density achieved, individuals mL−1) detected in Spirulina platensis cultures in covered raceway ponds under different media recycling conditions.

Amoeba Euplaesiobystra hypersalinica Nuclearia sp.

Group A

Group B

38

40

Group C

12

10

24

A, B

0.01

2.6. Statistical analysis

Ciliates Cyclidium glaucoma Euplotes vannus Monodinium sp. Meseres sp. Oxyrrhismarina sp. Schmidingerothrix sp. Spathidium spathula Vorticella microstoma

94 14 1 10 0 2 11 0

66 4 22 83 1 1 5 1

235 1 9 66 1 1 0 1

B A R B A, B A R A, B

0.46 0.01 < 0.01 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Rotifers Brachionus plicatilis Cephalodella sp. Lecane inermis eggs

57 0 1 4

91 9 21 34

74 2 2 18

O O O –

0.19 < 0.01 < 0.01 0.04

Totalb 14

10

13

12

–

–

Dominant species of microzooplankton were defined by the Dominance Index, which was calculated using the following formula: Y = (ni/N) × fi, where Y is the Dominance Index for each species, ni represents abundance of the species targeted, N represents the abundance of all species, and fi is the occurrence frequency of the species researched [39]. According to Xu and Chen [39], Y ≥ 0.02 would indicate dominance in a community. The software Origin 8.5 was used to examine microalgal growth, the dynamics of the microzooplankton communities and the microbiological compositions in the Spirulina cultures. A heatmap of microzooplankton relative abundance was generated using HemI software (Heatmap Illustrator, version 1.0) [40], where each colour of the heatmap denotes the proportion of one species in the total microzooplankton abundance (the data were transformed by log10 and then the transformed data used to generate the heatmap). To illustrate the relationship between microzooplankton communities and environmental factors, detrended correspondence analysis (DCA) on the species data was employed to determine whether canonical correspondence analysis (CCA, unimodal ordination) or redundancy analysis (RDA, linear ordination) should be applied first [41]. Among the ordination methods, the linear methods (RDA) reflect the Euclidean distances between the samples. Ordination analysis was performed using CANOCO version 4.5 (Biometrics Wageningen, The Netherlands) [41].

Groups of ponds (A, B and C) are as described in Tables 1. a Microzooplankton functional groups: A, algivores; B, bactivores−detrivores; O, omnivores; R, predators or raptors. Classification based on Wang [34], Shen [36] and our observation. Y, dominance index for each species (values of Y > 0.02 indicate that the species can be regarded as a dominant species); −, no data. b Excluding eggs.

2.4.2. Abundance analysis The densities of microzooplankton were determined by microscopic enumeration according to the method of Zhang and Huang [37]. Samples (50 mL) of algal culture were preserved with Lugols' iodine (1% final concentration) and shaken well before quantitative enumeration. An upright microscope (Olympus CX-31) was used to count the microzooplankton in the sample. Protozoa and rotifers were counted using a 0.10 mL and 1.0 mL phytoplankton counting chamber (CC-F, China), respectively, with two counts being made for each sample and the mean abundance for each sample calculated.

3. Results 3.1. Environmental factors during the cultivation Values of water temperature at the end of the experiment (29.0–30.1 °C), pH (9.50–9.85), conductivity (7.9 × 103–8.6 × 103 μs cm−1), salinity (4.0–4.64‰) and NOX concentrations (73 mg L−1 − 83.5 mg L−1) were comparable in the three different pond groups (Table 1). However, the content of dissolved organic carbon (DOC) in group A (17.6 mg L−1) was lower than in the other two groups (46 mg L−1 and 42 mg L−1 at groups B and C, respectively). Conversely, the content of dissolved oxygen (DO) and PO43−–P in group A was higher than in the other two groups. The concentration of NH4+–N was comparable in all three of the groups (11.9, 8.37 and 16.0 mg L−1 for A, B and C, respectively).

2.5. Amplicon sequencing of the prokaryotic contaminants The compositions of prokaryotic contaminants were evaluated with amplicon sequencing for the three groups of Spirulina cultures. A 10 mL sample of algal culture was collected from each pond on day 3 of the cultivation period, this representing the stationary phase of growth. There are two duplicates for each sample. The samples were filtered through 0.22 μm Millipore membranes, stored at −80 °C, and then subjected to total genomic DNA extraction with the PowerWater DNA isolation kit (MoBio, San Diego, USA) following the manufacturer's protocol. DNA concentrations were then determined using a NanoDrop 8000 UV spectrophotometer (Thermo Scientific, Massachusetts, USA). DNA was stored at −80 °C for subsequent analysis. The primer pair 517F/909R was used to amplify the V4-V5 region of the 16S rDNA gene [38]. PCR amplification was performed in duplicate using 1 μL genomic DNA template (1:10 dilution) in 25 μL reactions, containing 12.5 μL high fidelity 2 × PCR master mix (NEB, USA). The PCR procedure included an initial denaturation at 95 °C for 2 min, then 25 cycles of denaturation at 95 °C for 1 min, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension of 10 min at 72 °C. PCR products and negative controls were electrophoresed on a 1% (m/v) agarose gel. All the target bands of PCR product were purified using

3.2. The growth status of Spirulina under different media recycling conditions The growth status of Spirulina at the end of the experiment differed between experimental groups (Fig. 2 and Table 2). In terms of morphology, the algae in group A (Fig. 2A–B) were larger, more robust and more elastic, while the algae in groups B and C (Fig. 2C and D, respectively) were smaller and had less elasticity. Correspondingly, it was found that even though the cultures from all groups had a similar initial DW (below 0.40 g L−1), the algal growth trends were different between groups (Fig. 3). Cultures in group A kept growing and obtained the highest DW (2.8 g L−1) on the final day of the experiment, while cultures in group B and C reached their maximum DW (around 0.50 g L−1) the day before and then decreased to 0.30 g L−1 on the final day. The maximum biomass in group A was almost 5 times higher than that in 4

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Fig. 4. Differential interference contrast (DIC) micrographs of the main contaminating microzooplankton in Spirulina platensis cultures cultivated in raceway ponds. A: Euplaesiobystra hypersalinica; B, C: Nuclearia sp.; D: Monodinium sp.; E: Meseres sp.; F: Vorticella microstoma; G: Cyclidium glaucoma; H: Spathidium spathula; I–J: Euplotes vannus; K: Schmidingerothrix sp.; L: egg; M: Cephalodella sp.; N, O: Brachionus plicatilis; P: Lecane inermis. Scale bar: 10 μm for C; 40 μm for M, N and O; otherwise 20 μm.

the other two groups.

hypersalinica (Fig. 4A), Cyclidium glaucoma (Fig. 4G), and Brachionus plicatilis (Fig. 4N) were the dominant species as they occurred during the whole period of cultivation and the corresponding dominance index Y was > 0.02 (Table 3). Moreover, microscope observation indicated that B. plicatilis and E. hypersalinica were the most harmful contaminants for Spirulina culture as they were observed to graze heavily upon Spirulina. Some species, such as Cephalodella sp., Lecane inermis and Nuclearia sp., were observed to feed extensively upon Spirulina once they occurred but their abundance was very low (Table 3). Some other species, including Euplotes vannus, Schmidingerothrix sp., Vorticella microstoma and Oxyrrhismarina sp., are algivores but were rarely found to prey upon Spirulina cells. Some species, such as Cyclidium glaucoma and Meseres sp. (Table 3), are bactivores and cannot reduce algal biomass directly.

3.3. Composition and population dynamics of microzooplankton 3.3.1. Composition of microzooplankton under different media recycling conditions A total of 13 species of microzooplankton were identified in this study through morphological observation (Table 3 and Fig. 4). More species of microzooplankton were found in groups B and C (13 and 12 species respectively) than group A (10 species). Cephalodella sp., Vorticella microstoma and Oxyrrhismarina sp. were not found in group A during the whole period of investigation, while Spathidium spathula was not found in group C. Of these microzooplankton contaminants, Euplaesiobystra 5

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the three cultivation groups. Total microzooplankton abundance on the initial day was higher in groups B (237 individuals (inds.) mL−1) and C (131 inds. mL−1) than in group A (39 inds. mL−1) (Fig. 5). Microzooplankton abundance in group A kept increasing from the initial day and was highest on the final day (237 inds. mL−1) (Fig. 5A). The abundance of microzooplankton in group B reached its peak value (274 inds. mL−1) on day 2 and declined to the lowest value (118 inds. mL−1) on day 4, and then recovered to reach another peak value (274 inds. mL−1) on the final day (Fig. 5 B). The abundance of microzooplankton in group C kept increasing from day 1 to day 3, reaching a maximum of 409 inds. mL−1, then decreased to 107 inds. mL−1 on the final day (Fig. 5C). The heatmap of microzooplankton communities (Fig. 6) showed that the three dominant species – B. plicatilis, C. glaucoma and E. hypersalinica – were detected in all three pond groups throughout the whole cultivation period. However, there were differences in the occurrence of microzooplankton species between the groups. In group A, five species with low ratio were detected on the first day. These species existed throughout the cultivation period. As the cultivation progressed, four ciliates and one rotifer appeared in the cultures at low abundance and some species, such as Monodinium sp. and Lecane inermis, appeared occasionally. At the end of the Spirulina cultivation, there were eight species detected in the culture system. In group B, nine species were detected on the first day, and these species existed all the time except for Meseres sp., Schmidingerothrix sp. and Cephalodella sp. Three ciliates and one rotifer appeared during the cultivation. There were twelve species detected on the last day and the numbers and abundance of microzooplankton species were higher than in the other two groups. In group C, nine species appeared on the first day, but during the whole cultivation, the abundance of several species gradually decreased or even disappeared, so only seven species were found on the last day. 3.4. Composition of prokaryotic contaminants in Spirulina cultures Amplicon sequence data for environmental 16S rRNA gene sequences in our study are available on national center for biotechnology information (NCBI), and the sequence read archive (SRA) database accession number is SRP133463. Based on BLAST searches, sequences showed a similarity of 91–99% to environmental 16S rRNA gene sequences in the database (Table S1). A total of 90,982 DNA sequences (relative abundance above 0.01%) were found in the three groups. The heterotrophic bacterial sequences belonged to 42 operational taxonomic units (OTUs), including 34 different identified taxa (e.g., phyla, order, families, or genera) and 8 super OTUs which were the sum of phyla (Fig. 7). The most commonly found non-Cyanobacteria OTUs were related to Proteobacteria (35.71%), followed by other Bacteria (26.19%), Bacteroidetes (21.43%), Archaea (7.14%) and Firmicutes (4.76%). In terms of the dominant prokaryotic OTUs in the different pond groups, the most common OTU in groups A and C was Cyanobacteria, with relative abundances as high as 50%; however, in group B, Flavobacterium was the dominant OTU, with a relative abundance of 24.83%, and the relative abundance of Cyanobacteria was only 4% (Fig. 7). In group A, as well as Cyanobacteria the other two dominant OTUs were “other Proteobacteria” (6.82%) and “other Bacteroidetes” (6.29%). In group B, Flavobacterium (24.83%), Aliidiomarina (18.73%) and other Bacteroidetes (7.92%) were the top three dominant OTUs. In group C, as well as Cyanobacteria, Flavobacterium (12.13%) and unknown Bacteria (5.56%) were the other two dominant OTUs.

Fig. 5. Total microzooplankton and dominant species abundances in Spirulina platensis cultures over the time course of cultivation in raceway ponds. A, B and C, experimental groups of ponds are as described in Table 1. Each bar was the mean value of microzooplankton abundances generated from two or three experimental ponds.

3.5. The relationship between Spirulina/microzooplankton and environmental factors

3.3.2. Population dynamics of microzooplankton under different media recycling conditions The population dynamics of the microzooplankton differed between

The results of DCA based on species data showed that the longest gradient was lower than 4, so the RDA was chosen for analyzing the relationship between plankton (dominant microzooplankton species 6

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Fig. 6. Heatmap view of microzooplankton ratio (relative abundance) under different media recycling conditions over the time course of cultivation. Each column corresponds to a single microzooplankton taxon and each row corresponds to one day. White indicates that the microzooplankton taxon was not detected. The relative abundance of each taxon is along a grayscale from slight (light gray) to abundant (dark black).

abundance, total microzooplankton abundance and Spirulina biomass) and environmental factors (Fig. 8). The RDA explained 42% of the microzooplankton variation in the first two axes, and 96% of the species–environmental relationship. Samples from the three pond groups could be separated well into two parts, one part containing samples from group A and the other part containing samples from groups B and C. The high biomass of Spirulina in group A was related to the high phosphorus (PO43−–P) content of the culture system, with higher water temperatures and DO concentrations also being found in this group. The high densities of microzooplankton contaminants (dominant species and total microzooplankton) found in groups B and C were associated with high DOC concentration.

that can live in hypersaline or high temperature environments [43]; although there are only a few reports on its feeding habits, we observed substantial quantities of Spirulina within cells of E. hypersalinica. In our study, a greater abundance of harmful species was found in groups B and C with recycled medium (through redundancy analysis, Fig. 8), consistent with the previous view that cultures with recycled medium are more susceptible to contamination by predator contaminants [9]. In terms of the prokaryotic contaminant composition, Cyanobacteria was very obviously in the majority in our study, accounting for about 50% of relative abundance in pond groups A and C; however, it is not clear why the abundance of Cyanobacteria detected in group B was so low (only 4%). This phenomenon may be related to DNA extraction methods [44], as the inadequacy of DNA extraction methods may lead to the loss of information on some species. Flavobacterium, which was found to be the most abundant prokaryotic taxon in group B and the second most abundant in C but only accounted for 1.89% of relative abundance in group A, can cause health issues [25] and it is possible that the bacteria accumulate during the recycling of the medium. The other major prokaryotes in our ponds – Proteobacteria, Bacteroidetes and Archaea – are usually found in wastewater treatment systems (Proteobacteria), aquatic (Bacteroidetes) and terrestrial habitats (Archaea) [25,45]. Moreover, some of the OTUs detected were closely related to animal and human skin and gut (e.g., Firmicutes, Proteobacteria) [46]. This may indicate that animals that are attracted to water (e.g., birds, rodents) and humans handling the product during the different processing steps (e.g., harvesting, drying) may be sources of microbial contamination.

4. Discussion 4.1. The species composition of bio-contaminants under different media recycling conditions Shimamatsu [23] found that contamination is a common problem in mass production of Spirulina under conditions of continuous recycling of the culture medium. The results of the present study are consistent with this. The combination of morphological observation and amplicon sequencing allowed us to achieve more detailed information on the biodiversity in the Spirulina cultures than would have been possible otherwise, revealing a total of 13 species of ciliates, amoebae and rotifers (Table 3) and 42 prokaryotic OTUs (Fig. 7). The greater number of microzooplankton species in groups B and C (with recycled medium) than group A (with fresh medium) was possibly related to the higher DOC content in the group B and C ponds, as more organic matter can support more microzooplankton [35]. The observation that the microzooplanktonic species B. plicatilis and E. hypersalinica were the most harmful contaminants for Spirulina culture, as a result of their dominance, frequent occurrence and heavy grazing on Spirulina, is also consistent with reports in the literature about the damage these two species can cause to microalgae. B. plicatilis can feed on several kinds of microalgae and has high consumption rates on Arthrospira fusiformis [21,42]. E. hypersalinica is a eurytopic species

4.2. The population dynamic of microzooplankton under different media recycling conditions The abundance of microzooplankton changed differently in different media during the cultivation of Spirulina. The greater number of species found in groups B and C on the first day, compared to group A (Fig. 6), indicated that the use of recycled medium introduces more contaminants into the culture, this being consistent with other reports in the literature [23]. As cultures in group A were cultivated with fresh 7

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Fig. 7. Profile of prokaryotic taxa based on 16S rDNA gene sequences inferred from 42 biological contaminant operational taxonomic units (OTUs) with a relative abundance above 0.01%. Samples taken on day 3 of Spirulina platensis cultivation. Each bar was generated following DNA extraction of two biological replicates that was subject to Illumina Miseq platform with the PE300 program (are described in Materials and methods). OTUs comprised 34 different identified taxa (e.g., phyla, order, families, or genera) and 8 OTUs that were sum of phyla.

medium, the five contaminants in group A are likely to have been introduced via the algal seed cultures. Moreover, it was found that both the species number and abundances of microzooplankton in group B were higher than those in group C in the late stage of cultivation (Fig. 6), indicating that the culture conditions for group B provided a more favorable environment for microzooplankton growth. For group C, the reason for the decrease in the species abundance (Fig. 5) and number (Fig. 6) of microzooplankton may be that multiple cycles of recycling the medium had resulted in the production of inhibitors as reported by other researchers [12]. Some algae, such as Fischerella muscicola and Nodularia harveyana, have been reported to release allelopathic substances against algae and Cyanobacteria, bacteria and fungi that inhibit their growth or kill them [47–50]. It can be concluded that the there was a complex relationship between the extent of medium recycling and the species compositions and population dynamics of contaminating microzooplankton in the algal

cultures. More species of contaminants were introduced into cultures with recycled medium. And the more the culture medium was recycled, the more inhibitors were produced in the medium, which then inhibited the growth of microzooplankton. 4.3. The impact of recycled medium on algal growth Culture conditions are a very important influence on the growth of Spirulina in cultivation. In the present study, the environmental factors accounting for most of the variation between different culture conditions were dissolved organic carbon (DOC), dissolved oxygen (DO) and PO43−−P. The nutrient composition of the medium affects important physiological and biochemical processes that in turn affect productivity and the quality of the final product. Our finding that high biomass of Spirulina was related to high phosphorus content (Fig. 8) is consistent with the report by Markou and Georgakakis [51] that phosphorus is one 8

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doi.org/10.1016/j.algal.2019.101718. Author contributions All authors contributed to the conception and design of the article. Danni Yuan performed experiments, data collection and analysis, and wrote the article. Mimi Yao analyzed molecular data and built the figures. Lan Wang and Yanhua Li took part in data collection and management. Yingchun Gong and Qiang Hu reviewed and revised all versions of the article. The authors thank Fan Xiong from the Institute of Hydrobiology, Chinese Academy of Sciences, for her help in amplicon sequencing analysis. Declaration of competing interest Fig. 8. Redundancy analysis (RDA) of the relationship between plankton (Spirulina biomass and microzooplankton abundance) and environmental factors. Significant environmental variables are indicated by red arrows and Spirulina/microzooplankton by black arrows. Samples were taken on each day of cultivation and are represented with a symbol for the corresponding pond group. Brac, Brachionus plicatilis; EuplH, Euplaesiobystra hypersalinica; Cycli, Cyclidium marinum; TZ, total microzooplankton; WT, water temperature; DO, dissolved oxygen; DOC, dissolved organic carbon; NOxeN, concentrations of NO2−, NO3−; P, PO43–P. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The authors declare no conflict of interest. Acknowledgments This work was funded by National Key Research and Development Program of China (2018YFD0901504), National Natural Science Foundation of China (31772419, 31872201), SDIC Biotech Investment Co., Ltd., State Development and Investment Corporation, China (Y841171Z02), and the Chinese Academy of Sciences (ZDRW-ZS-20172-2).

of the basic nutrients required by microalgae and that it is important for microalgal growth. High Spirulina biomass in our study was also associated with a high concentration of DO (Fig. 8), high DO content indicating that the microalgae are in good physiological condition [9]. Microalgae will release oxygen through photosynthesis and it is very important, in outdoor Spirulina mass culture, to maintain high oxygenation if biomass production is to be optimized [9]. Both the high phosphorus and high DO conditions in our study were found in the group A ponds, with fresh medium. In contrast, the high content of DOC detected in groups B and C with lower Spirulina biomass reflect a greater abundance of bacteria in these cultures using recycled medium, as the DOC represents the production of the heterotrophic planktonic bacteria [52]. These results are consistent with those of Gast [53] who stated that more bacteria will appear with recycled medium. Our results showed, therefore, that recycling of the medium inhibits the production of Spirulina.

Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable. References [1] X.W. Zhang, Y.M. Zhang, F. Chen, Application of mathematical models to the determination optimal glucose concentration and light intensity for mixotrophic culture of Spirulina platensis, Process Biochem. 34 (1999) 477–481. [2] B. Gami, A. Naik, B. Patel, Cultivation of Spirulina species in different liquid media, J. Algal Biomass Utln. 2 (2011) 15–26. [3] A. Converti, A. Lodi, A.D. Borghi, C. Solisio, Cultivation of Spirulina platensis in a combined airlift-tubular reactor system, Biochem. Eng. J. 32 (2006) 13–18. [4] J. Leonard, P. Compere, Spirulina platensis Geitler, algue bleue de grande valeur alimentaire par sa richesse en proteines, Bull Jard Bot Nat Belg 37 (1967) 1. [5] D.M. Li, Y.Z. Qi, Spirulina industry in China: present status and future prospects, J. Appl. Phycol. 9 (1997) 25–28. [6] B.T. Wu, W.Z. Xiang, C.K. Tseng, Spirulina cultivation in China, Chin. J. Oceanol. Limnol. 16 (1998) 152–157. [7] J. Chen, Y. Wang, J.R. Benemann, X.C. Zhang, H.J. Hu, S. Qin, Microalgal industry in China: challenges and prospects, J. Appl. Phycol. 28 (2016) 715–725. [8] G.M.D. Rosa, L. Moraes, B.B. Cardias, M.R.A.Z. Souza, J.A.V. Costa, Chemical absorption and CO2 biofixation via the cultivation of Spirulina in semicontinuous mode with nutrient recycle, Bioresour. Technol. 192 (2015) 321–327. [9] A. Belay, Biology and industrial production of Arthrospira (Spirulina), Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second edition, John Wiley & Sons, Ltd, 2013, pp. 339–358. [10] S.H. Ho, W.B. Lu, J.S. Chang, Photobioreactor strategies for improving the CO2 fixation efficiency of indigenous Scenedesmus obliquus CNW-N: statistical optimization of CO2 feeding, illumination and operation mode, Bioresour. Technol. 105 (2012) 106–113. [11] J. Yang, M. Xu, X. Zhang, Q. Hu, M. Sommerfeld, Y. Chen, Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance, Bioresour. Technol. 102 (2011) 159–165. [12] A. Juneja, R. Ceballos, G. Murthy, Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review, Energiese 6 (2013) 4607–4638. [13] H. Wang, W. Zhang, L. Chen, J. Wang, T. Liu, The contamination and control of biological pollutants in mass cultivation of microalgae, Bioresour. Technol. 128 (2013) 745–750. [14] J. Gutman, A. Zarka, S. Boussiba, The host range of Paraphysoderma sedebokerensis, a chytrid that infects Haematococcus pluvialis, Eur. J. Phycol. 44 (2009) 509–514. [15] J.G. Day, Y. Gong, Q. Hu, Microzooplanktonic grazers – a potentially devastating threat to the commercial success of microalgal mass culture, Algal Res. 27 (2017) 356–365. [16] M.Y. Ma, D.N. Yuan, Y. He, M. Park, Y.C. Gong, Q. Hu, Effective control of Poterioochromonas malhamensis in massive culture of Chlorella sorokiniana GT-1 by maintaining CO2-mediated low culture pH, Algal Res. 26 (2017) 436–444. [17] E. Ganuza, C.E. Sellers, B.W. Bennett, E.M. Lyons, L.T. Carney, A novel treatment protects Chlorella at commercial scale from the predatory bacterium Vampirovibrio

5. Conclusion In mass production of Spirulina in open ponds, cultivation conditions affect the dynamics of microzooplankton communities and nutrient composition during the cultivation that in turn affects productivity and the quality of the final product. High algal biomass and fewer contaminant species will be achieved in the Spirulina culture if fresh medium is used, while we detected low algal biomass and higher microzooplankton biomass in Spirulina culture with recycled medium. Brachionus plicatilis and Euplaesiobystra hypersalinica are the main microzooplankton that are harmful to Spirulina growth. The use of recycled medium can not only limit the growth of Spirulina but also inhibit the growth of microzooplankton. It is difficult to conclude, however, that the bio-contaminants will directly affect the growth of Spirulina, which may also be co-affected by physico-chemical factors. Microzooplankton species number and abundance decreased in the late cultivation of Spirulina cultures with recycled medium, but microzooplankton abundance increased slowly during Spirulina cultivation with fresh medium. In the future, more research should be conducted to identify the critical biological and non-biological factors affecting the growth of Spirulina during cultivation in recycled medium, so that we can find the solution to reduce the negative effects of recycled medium. Supplementary data to this article can be found online at https:// 9

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