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Continuous cultivation of Arthrospira platensis for phycocyanin production in large-scale outdoor raceway ponds using microfiltered culture medium
Bioresource Technology 287 (2019) 121420
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Continuous cultivation of Arthrospira platensis for phycocyanin production in large-scale outdoor raceway ponds using microfiltered culture medium
T
Jiajia Yua, Hancui Hua, Xiaodan Wua, Congchun Wanga, Ting Zhoua, Yuhuan Liua, Roger Ruana,b, ⁎ Hongli Zhenga, a MOE Biomass Energy Research Center and College of Food Science and Technology and State Key Laboratory of Food Science and Technology, Nanchang University, 235 East Nanjing Road, Nanchang, Jiangxi 330047, People’s Republic of China b Center for Biorefining and Department of Bioproducts and Biosystems Engineering , University of Minnesota, 1390 Eckles Avenue, St. Paul, MN 55108, United States
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalga Microbiological contamination Phycocyanin production Microbial community Microfiltration
In the present study, the effect of filtrating algal culture medium for reuse by using microfiltration membranes on microalgal growth, microbiological contamination, and phycocyanin production of Arthrospira platensis was investigated. Results showed that microfiltered culture medium affected microalgal growth, microbiological contamination, and phycocyanin production of A. platensis significantly. Microfiltered culture medium could enhance biomass production, photosynthesis, and phycocyanin accumulation and decrease microbiological contamination during continuous cultivation of A. platensis compared to the control. The profile of microbial communities, which contained the 10 phyla of microorganisms including bacteria and microzooplanktons, was identified for the first time for industrial algae systems of A. platensis with extreme conditions (salt-alkaline stress conditions). The application of the established strategy can enhance phycocyanin production of A. platensis while mitigating microbiological contamination.
1. Introduction As one kinds of the microalgal species that have been produced on a large scale, Spirulina is widely used for the production of nutraceuticals,
medicine, aquaculture feed, poultry feed, and cosmetics (Markou and Nerantzis, 2013). Especially, one of major protein constituents, phycocyanin, found in Spirulina has been used as anticancer drug, antioxidant, antiviral drug, a potent therapeutic agent protecting a variety
⁎ Corresponding author at: College of Food Science and Technology, Nanchang University, 235 East Nanjing Road, Nanchang, Jiangxi 330047, People’s Republic of China. E-mail address: [email protected] (H. Zheng).
https://doi.org/10.1016/j.biortech.2019.121420 Received 12 March 2019; Received in revised form 1 May 2019; Accepted 2 May 2019 Available online 10 May 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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irradiance surface area of 100 m2 (about 33.3 m in length and 3 m in width), and the initial culture depth was approximately 30 cm at Ordos Lvfuyuan Biotechnology Corp. Ltd. (Ordos, Inner Mongolia Autonomous Region, China) from July 1 to 31, 2018. Prior to inoculation into the outdoor raceway ponds, microalgal cells were harvested by using 37-μm opening size sieves purchased from Nanchang Pinghai Biotechnology Development Co., Ltd. (Jiangxi Province, China) at room temperature and washed with distilled water for three times. In all cases, microalga was inoculated at 1:5 (v/v) ratio into the outdoor raceway ponds. The strain A. platensis was cultivated in the medium, which was composed of: 6, 000 g m−3 natural sodium bicarbonate (from the nearby alkaline lakes), 500 g m−3 KCl, 30 g m−3 MgSO4·7H2O, 10 g m−3 FeSO4·7H2O, and 30 g m−3 KH2PO4. The natural sodium bicarbonate was obtained by sun drying of the water from the nearby alkaline lakes. This crude natural soda which contained > 50% (w/w) of sodium bicarbonate was applied in the medium for A. platensis cultivation. A. platensis was grown in continuous cultivation. The biomass of A. platensis was harvested for the first time on day 5. After that, every two days one half of the algal suspension was harvested and the medium from the harvesting process was back to the raceway ponds. Due to some water loss through harvest and evaporation during cultivation, water was added to the raceway ponds to keep the volume constant at 30 m3 during cultivation process. At each harvest date, the feeding substrate was added into the raceway ponds with 160 g m−3 KCl, 10 g m−3 MgSO4·7H2O, 3 g m−3 FeSO4·7H2O, and 10 g m−3 KH2PO4. The cultivation cycle was 31 days. For microfiltration experiments, on day 21 the residual medium from algae harvesting process was treated by using microfiltration membranes to remove microalgal contaminants and microalgal pieces. After that, the filtrated culture medium was returned to the outdoor raceway ponds and reused. The microfiltration system (TMB-MF-55, nominal pore size: 0.1 µm, effective area: 20 m2) used in this study was provided by Tianbang National Engineering Research Center of Membrane Technology Co., Ltd. (Dalian, Liaoning Province, China). The microfiltration system was a filtration tank, which consisted of a microfiltration cell, a peristaltic pump, a vacuum meter, a pressure balance meter, a programmable logic controller, and several accessories. The microfiltration membranes were made of polystyrene. The capacity of the microfiltration system was approximately 5 m3 hr−1. Prior to the microfiltration experiment, Milli-Q water was filtered through the system to remove organic materials for 2 h. There was about 0.15 g L−1 biomass (dry weight) in the medium used in the microfiltration experiment. The microfiltration conditions were as follows: temperature 25 °C, transmembrane pressure 1.8 bar, cross-flow 5.79 m s−1. The operational flux was 25 L m−2h−1. After each experiment, the microfiltration system was cleaned with 0.025 mol L−1 NaOH for 30 min at 50 °C. The culture cultivated in the reused culture medium without microfiltration was used as control.
of diseases and promotion of optimal health, natural colorants, and fluorescent marker (Liu et al., 2016; Manirafasha et al., 2016; Mittal et al., 2013). China is one of the major producers of Spirulina biomass in the world with an annual output of over 10,000 tons dry powders, which accounts for > 70% of the global supply (Yuan et al., 2018). At present, the most common method of cultivation of Spirulina at large scales in China is outdoor raceway ponds. One of the main drawbacks of cultivation of Spirulina in large-scale outdoor raceway ponds is contamination from strains of bacteria or other outside organisms, which could lead to sudden and massive death of microalgal cells (Mooij et al., 2015). In outdoor raceway ponds, the interaction between the air and liquid culture is helpful for CO2 input and releasing of excess-dissolved oxygen, but this would of course increase the chances of contamination from the air (Logan and Roanld, 2011). The biological pollutants including zooplankton, bacteria, fungus, other microalgae and virus, can significantly constrain the growth of microalgae (Lam et al., 2018). Yuan et al. (2018) report that Brachionus plicatilis is the frequent species of microzooplankton and is the most harmful to Spirulina culture in China. Microalgal contaminants, fungi decreases biomass productivity of Spirulina platensis cultivated in outdoor raceway ponds (Wang et al., 2013a). The phycocyanin production is highly dependent on microalgal growth of Spirulina (Manirafasha et al., 2018). Therefore, microalgal contaminants would influence the phycocyanin production of Spirulina. The cost of culture medium accounts for about 35% of total cost of the algal biomass production (Molina Grima et al., 2003). To reduce production cost of the continuous culture of Spirulina, the algal culture medium is usually reused. The culture medium without suitable treatments for reuse easily leads to the enrichment of microalgal contaminants. Microalgal contaminants could be removed by microfiltration membranes (Ma et al., 2014). Lebleu et al. (2009) report that the transfer of the bacteria through artificial membranes is triggered by the cell-wall flexibility when the pores are smaller in size than the bacterial cells. There is little information on the effect of filtrating culture medium for reuse by using microfiltration membranes on microbiological contamination in biomass cultivation of Spirulina for phycocyanin production in large-scale outdoor raceway ponds. The aim of this study was to investigate the effect of microfiltered culture medium on microalgal growth, microbiological contamination, and phycocyanin production of Spirulina, Arthrospira platensis (A. platensis). We further attempted to determine the biodiversity of microorganisms in commercial A. platensis farms and establish a corresponding cultivation strategy to mitigate microbiological contamination. Finally, the activity of photosystem II (PS II) indicating the photosynthetic performance of microalga was assessed. 2. Materials and methods 2.1. Materials
2.3. Analytical methods The analytical-reagent chemicals were purchased from Sigma Chemical Company (Shanghai, China).
2.3.1. Analysis of pH and biomass Sample pH was measured by using a pH meter (Delta 320, MettlerToledo, Greifensee, Switzerland). A. platensis was centrifuged at 3, 200 × g for 5 min, washed with distilled water for three times. After harvest, the microalgal pellets were lyophilized in a freeze drier (Model FD-1A-50, Beijing Boyikang Lab Instrument Co., Ltd., Beijing, China) for dry weight measurements. The biomass concentration (dry mass) of A. platensis (X, g L–1) was calculated using optical density (OD) measurements at 560 nm by a UV/VIS spectrophotometer (Lambda 25, PerkinElmer, Inc., USA) according to the following equation:
2.2. Microalgal strain and cultivation conditions Species of microalga, A. platensis was kindly provided by Ordos Lvfuyuan Biotechnology Corp. Ltd. (Ordos, Inner Mongolia Autonomous Region, China). The strain A. platensis was preserved in the SOT medium(Aikawa et al., 2012), which was composed of (mg L−1): NaHCO3 16, 800, NaNO3 2, 500, K2SO4 1, 000, NaCl 1, 000, MgSO4·7H2O 200, CaCl2·2H2O 40, K2HPO4 500, Na2EDTA 80, FeSO4·7H2O 10, and trace elements solution 1 mL L−1. The trace elements solution consisted of: ZnSO4·7H2O 222 mg L−1, H3BO3 2, 860 mg L−1, MnSO4·7H2O 2, 500 mg L−1, CuSO4·5H2O 79 mg L−1, and NaMoO4·2H2O 21 mg L−1. The culture system was 30 m3 open raceway ponds with an
X = 0.61 × OD560
(R2 = 0.99)
(1)
Each sample of 5 mL microalgal suspension was used to measure its OD. Samples were diluted for 10 times to give an absorbance in the range of 0.1–1.0. The optical density measurements were periodically 2
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2.3.5. Measurement of nutrients The samples of algal suspensions for analysis were first centrifuged at 4, 800 × g for 10 min and then the supernatants were collected and stored at 4 °C for further use. They were diluted for 20 times and analyzed for TN, and TP following the Hach DR 5000 Spectrophotometer Manual (Hach, 2008). The supernatants were filtrated using 0.45-μm membrane filters (Millipore Corporation, USA) and diluted for 5 times for analysis. The diluted solution of 0.5 mL was injected into a Multi N/ C3100 Analyzer (Analytik Jena AG, Jena, Germany) for the analysis of the dissolved organic carbon (DOC).
checked by gravimetry. Biomass productivity P (g m−2 d−1) was calculated according to the following equation:
P=
(X2 − X1 + Xh ) × V (t2 − t1) × A −1
(2) −1
where X1 (g L ) and X2 (g L ) are the microalgal cell concentrations at culture time t1 (day) and t2 (day), respectively. Xh is the dry weight of the harvested biomass between culture time t1and t2. V (m3) and A (m2) are cultivation volume and irradiance area, respectively.
2.3.6. Phycocyanin analysis A sample of A. platensis (60 mL) was filtered using the 37-μm opening size sieves, washed with distilled water for three times and the pellets were resuspended in 6 mL of 0.125 mol L−1 sodium phosphate salt solution containing 4 mmol L−1 NaN3, 2 mmol L−1 mercaptoethanol and 2 mmol L−1 EDTA to extract the phycocyanin. The mixture was disrupted using a 600 W ultrasonic cell disintegrator (GA92-IID, Wuxi Shangjia Biotechnology Co., Ltd, Jiangsu Province, China) for 30 s with 5 s intervals for a duration of 10 min followed by centrifugation at 4800 × g for 5 min. The phycocyanin concentration in the supernatants was determined spectrophotometrically by measuring the absorbance at 280, 620, and 652 nm, respectively. The phycocyanin concentration (PCC, g L–1) of A. platensis was calculated using OD according to the following equation (Bennett and Bogorad, 1973):
2.3.2. Measurement of photosynthesis The maximum quantum yield of PSΠ (Fv/Fm), a key parameter indicating photosynthetic performance, was measured using a pulse-amplitude-modulation fluorometer (Heinz Walz Gmbh, Effeltrich, Germany). Each sample of 3 mL microalgal suspension was acclimatized in the dark for 15 min prior to the measurements of the light response curves of photosynthesis. Light response curves were developed by using WinControl software v.3.2. The measurements were carried out in 4-mL cuvettes and the microalgal suspension was mixed during measurements using a magnetic stirrer. Fv/Fm was defined as:
Fv / Fm = (Fm − F0)/Fm
(3)
where F0 and Fm are the initial and maximum chlorophyll fluorescence, respectively. Fv represents the variation in chlorophyll fluorescence between Fm and F0.
PCC =
OD620 − 0.474 × OD652 5.34
(4)
2.3.3. DNA amplification, and high throughput DNA sequencing Samples were collected from the outdoor raceway ponds on day 1, 11, 21, and 31 for the treatment and control, respectively. The microbial genomic DNA was extracted by using a PowerSoil DNA extraction kit (MoBio Laboratories, CA, USA) according to the manufacturer’s recommendations and PCR amplification of the 16S rRNA (V4 region) was carried out. The primers of 515F (5′-GTGCCAGCMGCCGCGG TAA-3′) and 806R (5′-GGACTACHVGGGT-WTCTAAT-3′) were used to target the microbial and archaeal 16S rRNA genes. The PCR system was as follows, within 25 μL reaction system, 10 μL supermix, 0.25 μL of each primer, 1 μL template DNA, and 13.75 μL sterile MilliQ H2O. The PCR program included the steps as bellow: initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, elongation at 72 °C for 30 s, and final extension at 72 °C for 5 min. PCR products were measured by 1% (w/v) agarose-gel electrophoresis. Amplification reactions were done in triplicate, and PCR reaction products were pooled prior to sequencing by using an Illumina MiSeq platform HiSeq 2500 system (Illumina, San Diego, CA, USA) at the Novogene Biotechnology Co., Ltd (Beijing, China). The sequencing was performed, raw reads were obtained, and low-quality fragments were filtered using Mothur as described in previous studies (Kozich et al., 2013). The pyrosequencing procedure, statistical and bioinformatics analysis were conducted.
where OD620 and OD652 are the absorbance of the phycocyanin extracts at 620, and 652 nm, respectively. The phycocyanin purity ratio (PPR) was defined as follows (Walter et al., 2011):
2.3.4. Cell count To calculate the abundance of the microzooplankton, a hemocytometer was used to count the microzooplankton with a microscope (Leica DM 1000, Leica Microsystems (Shanghai) Trading Co., Ltd., China) directly. To enumerate microorganism (except microzooplankton and A. platensis) cell count of the samples, 25.0 mL of each microalgal samples was resuspended in 225 mL sterile saline solution (0.9%). And then they were blended evenly, subsequently diluted up to 10−4 using the sterile saline solution and blended evenly again. At last 1 mL of each diluted samples were plated on the nutrient agar plate, which was consisted of (weight/volume) beef extract (0.3%), peptone (0.5%), sodium chloride (0.5%), and agar (0.2%). The microorganisms were cultured at 37 °C for 48 h, subsequently counted.
3.1. Effect of microfiltered culture medium on cell growth
PPR =
OD620 OD280
(5)
where OD280 is the absorbance of the phycocyanin extracts at 280 nm. The phycocyanin content (PCT) was defined as dry weight ratio of extracted phycocyanin to biomass by using the equation according to Sharma et al. (2014):
PCT =
PCC × 100% X
(6)
where X is the biomass concentration, PCC is the phycocyanin concentration. 2.4. Data analysis In this work, all runs were carried out at random. All trials were performed in triplicate. Analysis of variance (ANOVA) of the data was carried out as described in a previous study (Zheng et al., 2012). 3. Results and discussion
Outdoor raceway ponds are the most common cultivation system for Spirulina in China. The main obstacle to the cultivation system of Spirulina in large-scale outdoor raceway ponds is contamination, which is caused by microalgal contaminants from air, or land during cultivation process, or harvesting process or medium reuse process. The effects of microalgal contaminants on microalgal growth were constraining the growth of microalgae, causing sudden and massive death of microalgal cells, and competing with each other for nutrients and spaces (Wang et al., 2013b). Microalgal contaminants in the medium could be removed by using microfiltration membranes (Ma et al., 2014). The effect of microfiltered culture medium on microalgal growth is shown in 3
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Table 1 The growth performance of A. platensis during continuous cultivation in large-scale outdoor raceway ponds. Experiment
Time (day)
Biomass concentration (g L−1)
P (g m−2 d−1)
TN (mg L−1)
TP (mg L−1)
DOC (mg L−1)
Control
1 11 21 31
0.21 0.62 0.57 0.45
± ± ± ±
0.00 0.01 0.03 0.02
5±0 34 ± 2 32 ± 1 25 ± 2
62.5 56.4 53.9 25.1
± ± ± ±
0.3 0.5 0.2 0.3
32.8 29.3 30.2 16.7
± ± ± ±
0.0 0.1 0.3 0.5
28.6 ± 0.0 91.7 ± 0.3 123.2 ± 0.3 36.5 ± 0.2
Treatment
1 11 21 31
0.22 0.64 0.59 0.57
± ± ± ±
0.00 0.03 0.02 0.01
6±0 36 ± 2 33 ± 1 32 ± 0
62.0 55.3 57.8 20.6
± ± ± ±
0.0 0.1 0.4 0.2
31.9 30.4 29.7 14.2
± ± ± ±
0.2 0.2 0.4 0.2
29.7 ± 0.1 96.6 ± 0.3 106.5 ± 0.2 58.3 ± 0.1
Data are shown as their replicate mean ± standard deviation. The same below.
increasing culture time for both the treatment and control during the initial 4 days. After that, at the harvesting days both the treatment and control had relative lower Fv/Fm than those of the next day, respectively. There were no significant differences (P > 0.05) in the Fv/Fm of the treatment and control during the initial 20 days. After that, the Fv/ Fm of the treatment and control had significant differences (P < 0.05). The Fv/Fm of the control decreased significantly (P < 0.05) at the late days of cultivation. The results indicated that the culture medium for reuse without microfiltration inhibited photosynthesis of A. platensis. The possible reason was that algicidal products from biological pollutants in the culture medium inhibited microalgal photosynthesis (Lam et al., 2018). The results also indicated that microfiltered culture medium was benefit to photosynthesis of A. platensis. Nutrients and pH of the culture were shifted by microfiltration to remove metabolites of algal pieces, and biological pollutants. Fv/Fm of the microalgae was affected by the factors such as nutrients, temperature, light, pH, and algal species (Zheng et al., 2018, 2019; Yao et al., 2016). Fv/Fm of the microalga of the treatment reached peak values of 0.744 on day 8 while it did so of 0.739 on day 12 for the control. During the initial 5 days, biomass concentration of the treatment increased along with increasing culture time (Fig. 1). There were no significant differences (P > 0.05) in biomass concentration of the treatment and control during the initial 20 days. After that, biomass concentration of the control decreased significantly (P < 0.05). Similar results were reported by Yuan et al. (2011). However, biomass concentration of the treatment decreased slightly. The highest biomass concentration and P of the treatment were 0.65 g L−1 and 37 g m−2 d−1, respectively, on day 5. The highest biomass concentration and P of the control were 0.63 g L−1 and 35 g m−2 d−1, respectively, on day 5. During the investigation, both TN and TP of the treatment and control decreased with increasing culture time. On day 31, biomass concentration and P of the treatment were 1.27 times and 1.28 times that of the control, respectively. The results showed that the microalgal growth of the control was inhibited compared with the treatment. The possible reason is that competition for available nutrients between alga and biological pollutants inhibited microalgal growth (Lam et al., 2018). Furthermore, some biological pollutants such as microzooplanktons, which could be removed by microfiltration, consumed algal cells and thus their presence inhibited microalgal growth. The effect of microfiltered culture medium on pH was investigated. Fig. 2 shows that pH values increased sharply during the initial 5 days for the treatment and control, respectively. Similar results were obtained by a previous study (Zhou et al., 2017). Possible reasons for the pH values increase may have been the consumption of nitrate (Bedekar et al., 2010), the transport of hydroxide ions to the outside of the cell through a reaction catalyzed by the enzyme carbonic anhydrase during the conversion of bicarbonate ions inside the cell to provide CO2 for the photosynthetic reaction and the activity of ribulose 1, 5-bisphosphate carboxylase which was significantly dependent on pH, increasing at higher pH. And ribulose 1, 5-bisphosphate carboxylase was present in the photosynthetic apparatus of the microalga, where the H+ ions were
Fig. 1. Photosynthetic performance and biomass accumulation of A. platensis during continuous cultivation in large-scale outdoor raceway ponds (Fv/Fm of the control (▲), Fv/Fm of the treatment (●); Biomass concentration of the control ( ), Biomass concentration of the treatment ( )).
Fig. 2. pH and exopolysaccharide variations of A. platensis during continuous cultivation in large-scale outdoor raceway ponds (pH of the control (▲), pH of the treatment (●); Exopolysaccharide concentration of the control ( ), Exopolysaccharide concentration of the treatment( )).
Table 1 and Figs. 1 and 2. The culture cultivated in the reused culture medium without microfiltration was used as control. Filtrating the medium by using microfiltration membranes could increase production cost of the continuous cultivation of Spirulina because of energy consumption, labor and equipment cost. Based on cost-benefit perspective, the culture medium for reuse during the whole cultivation process was only filtrated for one time on day 21. Fv/Fm from the treatment and control as a function of culture time was investigated. Fig. 1 shows that Fv/Fm increased along with 4
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3.2. Effect of microfiltered culture medium on microbiological contamination
sequestered to the inside of the tylacoid membrane and these light-induced energy fluxes resulted in an increase in pH values and activating the rubisco enzyme (Francisco et al., 2010). After that, pH values increased slightly for the treatment and control. The possible reason is that the supplement of sodium bicarbonate in the crude natural soda alleviated pH increase. Furthermore, pH of the culture medium was stabilized by exopolysaccharides (De Philippis et al., 2001), which were released by A. platensis (Fig. 2). pH values decreased significantly (P < 0.05) after day 20 for the control. It might be that microalgal contaminants of the control increased with increasing culture time and utilize exopolysaccharide to metabolize acidic substances leading to the pH drop. A lot of cyanobacteria were characterized by the presence of polysaccharidic outermost investments (De Philippis et al., 2001). Three kinds of investments including sheaths, capsules, and slimes were found in cyanobacteria. Most exopolysaccharides, which were released into the culture medium as water-soluble polymer, were in the existence of capsules and/or slimes (De Philippis and Vincenzini, 1998). And sheaths only accounted for a small fraction of the cell dry weight. Exopolysaccharides could trap metal ions, alter the rheological behaviour of water, and stabilize the flow properties of the culture medium under drastic variation of temperature, ionic strength and pH (De Philippis et al., 2001). Thus exopolysaccharides produced from microalga would affect itself growth. The effect of microfiltration of the reused culture medium on exopolysaccharide concentration was investigated. Exopolysaccharide concentrations increased from 0.24 mg L−1 to 2.31 mg L−1 for the treatment and from 0.25 mg L−1 to 2.26 mg L−1 for the control during the initial 20 days, respectively (Fig. 2). The exopolysaccharide concentrations for both the treatment and control were decreased greatly after that. DOC of the two cultures, which was caused partially by exopolysaccharides, reached their peaks before they decreased with increasing culture time (Table 1). The peaks of the DOC in the treatment and control were 106.5 mg L−1, and 123.2 mg L−1, respectively, on day 21. DOC decreased at the late days of cultivation with decreasing exopolysaccharide concentration. It may be that exopolysaccharides were composed of the hexoses glucose, galactose, mannose, pentoses ribose, arabinose, etc (De Philippis et al., 2001). Most of them could be used as substrates for microbial growth. Thus exopolysaccharide concentration decreased at the late days of cultivation with increasing the cell density of bacteria and microzooplankton (Table 2). The exopolysaccharide concentration of the treatment had a sharp decline on day 21. This is because that some exopolysaccharides in the culture medium for reuse were removed by microfiltration on day 20 causing the decrease of exopolysaccharide concentration. It indicated that microfiltration had significant effect (P < 0.05) on exopolysaccharide concentration.
To overcome the challenges of microbiological contamination, researchers have been searching for feasible approaches to control them. Previous studies preferred filtrating the culture medium of microalgae to eradicate the microbiological contamination to adding drugs to annihilate the microbiological contamination (Lam et al., 2018; Wang et al., 2013b). Microfiltered culture medium had significant effect (P < 0.05) on the amount of bacteria and microzooplanktons (Table 2). For both the treatment and control, the amount of bacteria and microzooplankton all increased with increasing culture time. A massive outbreak of bacteria and microzooplankton did not occur in this study. The cultures like Spirulina were a limited number of microalgae, which grew successfully in large-scale outdoor raceway ponds because of their extreme growth conditions such as very high salinity and high pH (Wang et al., 2013b). In the present study, natural sodium bicarbonate from the nearby alkaline lakes was applied in the medium for A. platensis cultivation. Therefore, A. platensis was grew under saltalkaline stress conditions. However, very high salinity and high pH decreased the growth of bacteria and microzooplanktons thus minimizing the chances of massive outbreak of bacteria and microzooplanktons (Lee, 2001). Furthermore, A. platensis used inorganic matters to synthesize complex molecules through photosynthesis to support its growth and multiplication. Most of bacteria and microzooplanktons needed organic matters to support their growth and multiplication. Therefore, the chances of massive outbreak of bacteria and microzooplanktons was further decreased due to the used medium in this study. Before day 11, no microzooplankton was observed in the cultures of the treatment and control (Table 2). Before day 21, both the amount of bacteria and microzooplanktons were no significant difference (P > 0.05) between the treatment and control. On day 31, the treatment and control had significant difference (P < 0.05) on both the amount of bacteria and microzooplanktons. Bacterial and microzooplankton taxa of the control on day 31 were 9.3 × 105 CFU L−1 and 1.5 × 104 inds. L−1, respectively, which were about 3 and 7 times that of the treatment, respectively. It indicated that microfiltered culture medium could decrease the amount of bacteria and microzooplanktons. Algal suspensions were collected over an One-month period from continuous algae cultivation systems in large-scale outdoor raceway ponds at Ordos Lvfuyuan Biotechnology Corp. Ltd. The algal suspensions were harvested by centrifugation at 9, 600 × g for 15 min. Community DNA was extracted from harvested biomass samples. In total, 24 samples were processed. The V4 region of 16S rRNA genes was amplified and sequenced, generating about 12 million sequenced amplicons. Following filtering steps that removed algae-derived chloroplast and mitochondrial sequences along with extremely rare sequences and other potential sources of error, 2 million microbial reads were used for further analyses. Microbial communities were characterized in the cultivation of A. platensis of the treatment and control. The composition of these microbial communities was compared across all samples using unweighted UniFrac as a distance metric. Algae cultivation of the treatment and control contained microbial communities that were distinct in terms of phylogenetic structure. The microbial community structures of the treatment and control were analyzed. Taxonomic distributions at the phyla level were summarized for the treatment and control samples (Fig. 3). At the phylum level, the ten most abundant phyla were Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia, Spirochaetes, Lentisphaerae, Deinococcus-Thermus, and Protozoa. They could grow under salt-alkaline stress conditions. The microbiological contaminants were zooplankton, bacteria, fungus, other microalgae and virus during microalgal cultivation in open ponds (Lam et al., 2018). The microbiological contaminants in this study only belonged to bacteria and microzooplanktons. At the phylum level,
Table 2 The cell density of bacteria and microzooplanktons. Experiment
Microorganism count
Time (day) 1
Control
Treatment
Bacterial taxa (CFU L−1) Microzooplankton taxa (inds. L−1) Bacterial taxa (CFU L−1) Microzooplankton taxa (inds. L−1)
11 3
21 4
31 5
9.3 × 105
2.0 × 10
2.6 × 10
3.9 × 10
0
0
2.5 × 103
1.5 × 104
1.7 × 103
3.5 × 104
5.1 × 105
2.8 × 105
0
0
3.0 × 103
2.2 × 103
5
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were Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia, Spirochaetes, Lentisphaerae, Deinococcus-Thermus, and Protozoa for both the treatment and control. It showed that new phyla were appeared with increasing culture time. Since the processes of cultures of the treatment and control were done under unsterile conditions in large-scale outdoor raceway ponds, each process, the initial algal inoculum or the harvesting process or the reused medium, was an opportunity for bacteria and other microbes to enter the community and increased species richness and phylogenetic diversity. Beyond those processes, there were numerous environmental differences during cultivation in large-scale outdoor raceway ponds that might affect microbial populations and cause distinct communities to dominate different cultivation systems. Some of these factors would directly influence microbes (e.g., pH, nutrients), while others (e.g., light intensity, oxygen) had impacts on the growth of A. platensis (Chen et al., 2013), which in turn would influence microbial growth. Filtrating culture medium could remove microbes, which consumed nutrients, and oxygen during their growth and their metabolites influencing medium pH. Once light entered medium, microbes as shading light materials limited the amount of available light for A. platensis. In addition to differences in environmental parameters, the continuous cultivation process may affect microbial community composition. In the continuous cultivation system used here, biomass from half of dense A. platensis cultures of the test was harvested and continuous cultures were not inoculated; occasionally, microbes was still in the reused medium. Because culture communities (A. platensis, microbes, and other constituents) were repeatedly reused for continuous cultivation, this cultivation strategy provided additional generations within which communities may have been affected by the conditions of that reused medium and therefore became increasingly distinct from communities grown with different reused medium.
Fig. 3. Microorganism abundance in phylum level. (PB: Proteobacteria; B: Bacteroidetes; F: Firmicutes; A: Actinobacteria; PL: Planctomycetes; V: Verrucomicrobia; S: Spirochaetes; L: Lentisphaerae; D: Deinococcus-Thermus; PT: Protozoa; O: Others.) (C1: control on day 1, C2: control on day 11, C3: control on day 21, C4: control on day 31; T1: treatment on day 1, T2: treatment on day 11, T3: treatment on day 21, T4: treatment on day 31).
Proteobacteria was the most abundant phylum in the community for all the tested samples. Proteobacteria and Bacteroidetes dominated communities from all the tested samples. On day 1, there were two phyla most abundant, Proteobacteria and Bacteroidetes, for the treatment and control accounted for 97%, and 97% of the microbial communities, respectively (Fig. 3). On day 31, the two most abundant phyla for the treatment and control were accounted for 56%, and 46% of the microbial communities, respectively. The results showed that the total abundance of Proteobacteria and Bacteroidetes was decreased with increasing culture time for the treatment and control, respectively. Considering all samples, Proteobacteria became less prevalent as culture time increased, having relative abundances of 56%, 49%, 39%, and 34% in cultivation grown on day 1, 11, 21, and 31 for the treatment, respectively and 57%, 55%, 40%, and 30% in cultivation grown on day 1, 11, 21, and 31 for the control, respectively. Bacteroidetes also decreased in relative abundance as culture time increased, from 41% abundance in the cultivation of the treatment on day 1 to 38% abundance in the cultivation of the treatment on day 11 to 32% abundance in the cultivation of the treatment on day 21 to 22% abundance in the cultivation of the treatment on day 31 and from 40% abundance in the cultivation of the control on day 1 to 33% abundance in the cultivation of the control on day 11 to 25% abundance in the cultivation of the control on day 21 to 16% abundance in the cultivation of the control on day 31 (Fig. 3). Within each cultivation method, microorganisms were ranked by relative abundance. On day 1, the two dominant phyla were Proteobacteria and Bacteroidetes for both the treatment and control (Fig. 3). Proteobacteria and Bacteroidetes previously have been shown to be the most abundant bacteria in algal cultivation system (Fulbright et al., 2018; Simek et al., 2011). On day 31, the ten dominant phyla
3.3. Effect of microfiltered culture medium on phycocyanin production of A. platensis Culture conditions such as culture medium composition, light intensities, temperature, pH, etc. had a significant impact on the microalgae‘s abilities to synthesize phycocyanin (Chen et al., 2013; Yao et al., 2016; Manirafasha et al., 2018). In order to identify the effect of microfiltered culture medium on phycocyanin production, A. platensis was continuously cultured in large-scale outdoor raceway ponds. The PCT was measured on day 1, 11, 21, and 31. The experimental results are presented in Table 3 and Fig. 4. The PCT on day 1 was much lower than that on day 11, 21, and 31 for the treatment and control, respectively. There was a decrease in PCT of the control when the culture time increased from day 11 to 31. The PCT of 10% of the treatment on day 31 was higher than that of 8% of the control. During the investigation, both PCT and protein content of the treatment and control increased before they decreased with increasing culture time. Similar results were obtained by Manirafasha et al. (2016). Phycocyanin was one of proteins found in A. platensis. The results showed that the variation of PCT was highly related to the change of protein content. The carbohydrate
Table 3 The phycocyanin production of A. platensis during continuous cultivation in large-scale outdoor raceway ponds. Experiment
Time (day)
Carbohydrate content (%)
Protein content (%)
PCC (mg L−1)
PCT (%)
PPR
Control
1 11 21 31
18 20 35 42
± ± ± ±
0 1 2 1
41 45 36 31
± ± ± ±
1 1 2 1
10.5 68.2 57.0 40.5
± ± ± ±
0.0 0.5 0.2 0.8
5±0 12 ± 1 11 ± 0 8±1
0.654 0.713 0.722 0.709
± ± ± ±
0.005 0.009 0.012 0.007
Treatment
1 11 21 31
19 21 32 35
± ± ± ±
0 1 1 2
41 49 43 34
± ± ± ±
0 3 2 1
13.2 76.8 64.9 68.4
± ± ± ±
0.4 0.6 0.7 0.3
6±0 14 ± 1 12 ± 0 10 ± 0
0.660 0.717 0.713 0.720
± ± ± ±
0.004 0.008 0.007 0.003
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Phycobiliprotein: potential microalgae derived pharmaceutical and biological reagent. Biochem. Eng. J. 109, 282–296. Manirafasha, E., Murwanashyaka, T., Ndikubwimana, T., Ahmeda, N.R., Liu, J.Y., Lu, Y.H., Zeng, X.H., Ling, X.P., Jing, K.J., 2018. Enhancement of cell growth and phycocyanin production in Arthrospira (Spirulina) platensis by metabolic stress and nitrate fed-batch. Bioresourc. Technol. 255, 293–301. Markou, G., Nerantzis, E., 2013. Microalgae for high-value compounds and biofuels production: a review with focus on cultivation under stress conditions. Biotechnol. Adv. 31, 1532–1542. Mittal, A.K., Chisti, Y., Banerjee, U.C., 2013. Synthesis of metallic nanoparticles using plantextracts. Biotechnol. Adv. 31 (2), 346–356. Molina Grima, E., Belarbi, E., Acien Fernandez, F., Robles Medina, A., Chisti, Y., 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 20, 491–515. Mooij, P.R., Stouten, G.R., van Loosdrecht, M.C.M., Kleerebezem, R., 2015. Ecology-based selective environments as solution to contamination in microalgal cultivation. Curr. Opin. Biotechnol. 33, 46–51. Sharma, G., Kumar, M., Ali, M., Jasuja, N., 2014. Effect of carbon content, salinity and pH on Spirulina platensis for phycocyanin, allophycocyanin and phycoerythrin accumulation. J. Microb. Biochem. Technol. 6, 202–206. Simek, K., Kasalicky, V., Zapomelova, E., Hornak, K., 2011. Alga-derived substrates select for distinct Betaproteobacterial lineages and contribute to niche separation in Limnohabitans strains. Appl. Environ. Microbiol. 77, 7307–7315. Walter, A., Carvalho, J.C.D., Soccol, V.T., Faria, A.B.B.D., Ghiggi, V., Soccol, C.R., 2011. Study of phycocyanin production from Spirulina platensis under different light spectra. Braz. Arch. Biol. Technol. 54 (4), 675–682. Wang, G., Fan, J., Wu, H., Zhang, X., Li, G., Zhang, H., Yang, X., Ye, F., Xiang, W., Li, X., 2013a. Nonhongiella spirulinensis, gen. nov. sp. nov. a bacterium isolated from a cultivation pond of Spirulina platensis, in Sanya, China. Anton. Leeuw. 106, 933–939. Wang, H., Zhang, W., Chen, L., Wang, J.F., Liu, T.Z., 2013b. The contamination and control of biological pollutants in mass cultivation of microalgae. Bioresourc. Technol. 128, 745–750. Yao, C.H., Pan, Y.F., Lu, H.B., Wu, P.C., Meng, Y.Y., Cao, X.P., Xue, S., 2016. Utilization of recovered nitrogen from hydrothermal carbonization process by Arthrospira platensis. Bioresour. Technol. 212, 26–34. Yuan, X., Kumar, A., Sahu, A.K., Ergas, S.J., 2011. Impact of ammonia concentration on Spirulina platensis growth in an airlift photobioreactor. Bioresour. Technol. 202, 3234–3239. Yuan, D.N., Zhan, X.L., Wang, M.Y., Wang, X.H., Feng, W.S., Gong, Y.C., Hu, Q., 2018. Biodiversity and distribution of microzooplankton in Spirulina (Arthrospira) platensis mass cultures throughout China. Algal Res. 30, 38–49. Zheng, H.L., Gao, Z., Yin, F.W., Ji, X.J., Huang, H., 2012. Effect of CO2 supply conditions
Fig. 4. Phycocyanin production of A. platensis during continuous cultivation in large-scale outdoor raceway ponds (C: control, T: treatment).
content of the treatment and control all increased along with increasing culture time. To a certain extent, there was a negative correlation between protein content and carbohydrate content. The PCC increased significantly (P < 0.05) during the initial 5 days for the treatment and control, respectively, and decreased slightly during the last 10 days for the control (Table 3 and Fig. 4). The PCC and PPR of the control all increased before they decreased along with increasing culture time. The PCC and PPR on day 1 was much lower than those on day 11, 21, and 31 for the treatment, respectively. The PCC and PPR of the treatment had little variation at the late 20 days, respectively. There was much difference in PCC and PPR on day 31 between the treatment and control. Especially, the highest PCC was 68.2 mg L−1 with a PPR of 0.713 for the control, while the highest PCC was 76.8 mg L−1 with a PPR of 0.717 for the treatment. The results herein presented attest that the hypothesis might be a reality. The PCT and PCC could be enhanced through filtrating the culture medium by using microfiltration membranes prior to reuse.
4. Conclusions A. platensis was able to produce phycocyanin efficiently in the existence of microbiological contamination in large-scale outdoor raceway ponds. Our study is the first report to identify the major harmful microbial species in commercial Spirulina farms. Ten phyla of microorganisms belonging to bacteria and microzooplanktons were identified. A strategy was established to alleviate microbiological contamination by using microfiltration membranes during continuous cultivation of A. platensis. Considering the environmental benefits and product quality, it is a promising way of using microfiltration, which could efficiently remove the microbiological contaminants, for the treatment of the culture medium for reuse subsequently.
Acknowledgements This work was supported in part by grants from the National Natural Science Foundation of China (Grant No. 21767017), and the Science and Technology Project of Jiangxi Provincial Department of Science and Technology, China (Grant Nos. 20171BBF60024 and 20181BBF60026), and the Innovation and Entrepreneurship Development Fund of ‘‘Thousand talents program” Talent of Jiangxi Province, China (Grant No. 1001-02102082).
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Zheng, H.L., Wan, X.D., Zou, G.Y., Zhou, T., Liu, Y.H., Ruan, R., 2019. Cultivation of Chlorella vulgaris in manure-free piggery wastewater with high-strength ammonium for nutrients removal and biomass production: effect of ammonium concentration, carbon/nitrogen ratio and pH. Bioresour. Technol. 273, 203–211. Zhou, W.Z., Li, Y.T., Gao, Y.Z., Zhao, H.X., 2017. Nutrients removal and recovery from saline wastewater by Spirulina platensis. Bioresourc. Technol. 245, 10–17.
on lipid production of Chlorella vulgaris from enzymatic hydrolysates of lipid-extracted microalgal biomass residues. Bioresour. Technol. 126, 24–30. Zheng, H.L., Liu, M.Z., Lu, Q., Wan, X.D., Ma, Y.W., Cheng, Y.L., Addy, M., Liu, Y.H., Ruan, R., 2018. Balancing carbon/nitrogen ratio to improve nutrients removal and algal biomass production in piggery and brewery wastewaters. Bioresour. Technol. 249, 479–486.
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Continuous cultivation of Arthrospira platensis for phycocyanin production in large-scale outdoor raceway ponds using microfiltered culture medium
T
Jiajia Yua, Hancui Hua, Xiaodan Wua, Congchun Wanga, Ting Zhoua, Yuhuan Liua, Roger Ruana,b, ⁎ Hongli Zhenga, a MOE Biomass Energy Research Center and College of Food Science and Technology and State Key Laboratory of Food Science and Technology, Nanchang University, 235 East Nanjing Road, Nanchang, Jiangxi 330047, People’s Republic of China b Center for Biorefining and Department of Bioproducts and Biosystems Engineering , University of Minnesota, 1390 Eckles Avenue, St. Paul, MN 55108, United States
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalga Microbiological contamination Phycocyanin production Microbial community Microfiltration
In the present study, the effect of filtrating algal culture medium for reuse by using microfiltration membranes on microalgal growth, microbiological contamination, and phycocyanin production of Arthrospira platensis was investigated. Results showed that microfiltered culture medium affected microalgal growth, microbiological contamination, and phycocyanin production of A. platensis significantly. Microfiltered culture medium could enhance biomass production, photosynthesis, and phycocyanin accumulation and decrease microbiological contamination during continuous cultivation of A. platensis compared to the control. The profile of microbial communities, which contained the 10 phyla of microorganisms including bacteria and microzooplanktons, was identified for the first time for industrial algae systems of A. platensis with extreme conditions (salt-alkaline stress conditions). The application of the established strategy can enhance phycocyanin production of A. platensis while mitigating microbiological contamination.
1. Introduction As one kinds of the microalgal species that have been produced on a large scale, Spirulina is widely used for the production of nutraceuticals,
medicine, aquaculture feed, poultry feed, and cosmetics (Markou and Nerantzis, 2013). Especially, one of major protein constituents, phycocyanin, found in Spirulina has been used as anticancer drug, antioxidant, antiviral drug, a potent therapeutic agent protecting a variety
⁎ Corresponding author at: College of Food Science and Technology, Nanchang University, 235 East Nanjing Road, Nanchang, Jiangxi 330047, People’s Republic of China. E-mail address: [email protected] (H. Zheng).
https://doi.org/10.1016/j.biortech.2019.121420 Received 12 March 2019; Received in revised form 1 May 2019; Accepted 2 May 2019 Available online 10 May 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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irradiance surface area of 100 m2 (about 33.3 m in length and 3 m in width), and the initial culture depth was approximately 30 cm at Ordos Lvfuyuan Biotechnology Corp. Ltd. (Ordos, Inner Mongolia Autonomous Region, China) from July 1 to 31, 2018. Prior to inoculation into the outdoor raceway ponds, microalgal cells were harvested by using 37-μm opening size sieves purchased from Nanchang Pinghai Biotechnology Development Co., Ltd. (Jiangxi Province, China) at room temperature and washed with distilled water for three times. In all cases, microalga was inoculated at 1:5 (v/v) ratio into the outdoor raceway ponds. The strain A. platensis was cultivated in the medium, which was composed of: 6, 000 g m−3 natural sodium bicarbonate (from the nearby alkaline lakes), 500 g m−3 KCl, 30 g m−3 MgSO4·7H2O, 10 g m−3 FeSO4·7H2O, and 30 g m−3 KH2PO4. The natural sodium bicarbonate was obtained by sun drying of the water from the nearby alkaline lakes. This crude natural soda which contained > 50% (w/w) of sodium bicarbonate was applied in the medium for A. platensis cultivation. A. platensis was grown in continuous cultivation. The biomass of A. platensis was harvested for the first time on day 5. After that, every two days one half of the algal suspension was harvested and the medium from the harvesting process was back to the raceway ponds. Due to some water loss through harvest and evaporation during cultivation, water was added to the raceway ponds to keep the volume constant at 30 m3 during cultivation process. At each harvest date, the feeding substrate was added into the raceway ponds with 160 g m−3 KCl, 10 g m−3 MgSO4·7H2O, 3 g m−3 FeSO4·7H2O, and 10 g m−3 KH2PO4. The cultivation cycle was 31 days. For microfiltration experiments, on day 21 the residual medium from algae harvesting process was treated by using microfiltration membranes to remove microalgal contaminants and microalgal pieces. After that, the filtrated culture medium was returned to the outdoor raceway ponds and reused. The microfiltration system (TMB-MF-55, nominal pore size: 0.1 µm, effective area: 20 m2) used in this study was provided by Tianbang National Engineering Research Center of Membrane Technology Co., Ltd. (Dalian, Liaoning Province, China). The microfiltration system was a filtration tank, which consisted of a microfiltration cell, a peristaltic pump, a vacuum meter, a pressure balance meter, a programmable logic controller, and several accessories. The microfiltration membranes were made of polystyrene. The capacity of the microfiltration system was approximately 5 m3 hr−1. Prior to the microfiltration experiment, Milli-Q water was filtered through the system to remove organic materials for 2 h. There was about 0.15 g L−1 biomass (dry weight) in the medium used in the microfiltration experiment. The microfiltration conditions were as follows: temperature 25 °C, transmembrane pressure 1.8 bar, cross-flow 5.79 m s−1. The operational flux was 25 L m−2h−1. After each experiment, the microfiltration system was cleaned with 0.025 mol L−1 NaOH for 30 min at 50 °C. The culture cultivated in the reused culture medium without microfiltration was used as control.
of diseases and promotion of optimal health, natural colorants, and fluorescent marker (Liu et al., 2016; Manirafasha et al., 2016; Mittal et al., 2013). China is one of the major producers of Spirulina biomass in the world with an annual output of over 10,000 tons dry powders, which accounts for > 70% of the global supply (Yuan et al., 2018). At present, the most common method of cultivation of Spirulina at large scales in China is outdoor raceway ponds. One of the main drawbacks of cultivation of Spirulina in large-scale outdoor raceway ponds is contamination from strains of bacteria or other outside organisms, which could lead to sudden and massive death of microalgal cells (Mooij et al., 2015). In outdoor raceway ponds, the interaction between the air and liquid culture is helpful for CO2 input and releasing of excess-dissolved oxygen, but this would of course increase the chances of contamination from the air (Logan and Roanld, 2011). The biological pollutants including zooplankton, bacteria, fungus, other microalgae and virus, can significantly constrain the growth of microalgae (Lam et al., 2018). Yuan et al. (2018) report that Brachionus plicatilis is the frequent species of microzooplankton and is the most harmful to Spirulina culture in China. Microalgal contaminants, fungi decreases biomass productivity of Spirulina platensis cultivated in outdoor raceway ponds (Wang et al., 2013a). The phycocyanin production is highly dependent on microalgal growth of Spirulina (Manirafasha et al., 2018). Therefore, microalgal contaminants would influence the phycocyanin production of Spirulina. The cost of culture medium accounts for about 35% of total cost of the algal biomass production (Molina Grima et al., 2003). To reduce production cost of the continuous culture of Spirulina, the algal culture medium is usually reused. The culture medium without suitable treatments for reuse easily leads to the enrichment of microalgal contaminants. Microalgal contaminants could be removed by microfiltration membranes (Ma et al., 2014). Lebleu et al. (2009) report that the transfer of the bacteria through artificial membranes is triggered by the cell-wall flexibility when the pores are smaller in size than the bacterial cells. There is little information on the effect of filtrating culture medium for reuse by using microfiltration membranes on microbiological contamination in biomass cultivation of Spirulina for phycocyanin production in large-scale outdoor raceway ponds. The aim of this study was to investigate the effect of microfiltered culture medium on microalgal growth, microbiological contamination, and phycocyanin production of Spirulina, Arthrospira platensis (A. platensis). We further attempted to determine the biodiversity of microorganisms in commercial A. platensis farms and establish a corresponding cultivation strategy to mitigate microbiological contamination. Finally, the activity of photosystem II (PS II) indicating the photosynthetic performance of microalga was assessed. 2. Materials and methods 2.1. Materials
2.3. Analytical methods The analytical-reagent chemicals were purchased from Sigma Chemical Company (Shanghai, China).
2.3.1. Analysis of pH and biomass Sample pH was measured by using a pH meter (Delta 320, MettlerToledo, Greifensee, Switzerland). A. platensis was centrifuged at 3, 200 × g for 5 min, washed with distilled water for three times. After harvest, the microalgal pellets were lyophilized in a freeze drier (Model FD-1A-50, Beijing Boyikang Lab Instrument Co., Ltd., Beijing, China) for dry weight measurements. The biomass concentration (dry mass) of A. platensis (X, g L–1) was calculated using optical density (OD) measurements at 560 nm by a UV/VIS spectrophotometer (Lambda 25, PerkinElmer, Inc., USA) according to the following equation:
2.2. Microalgal strain and cultivation conditions Species of microalga, A. platensis was kindly provided by Ordos Lvfuyuan Biotechnology Corp. Ltd. (Ordos, Inner Mongolia Autonomous Region, China). The strain A. platensis was preserved in the SOT medium(Aikawa et al., 2012), which was composed of (mg L−1): NaHCO3 16, 800, NaNO3 2, 500, K2SO4 1, 000, NaCl 1, 000, MgSO4·7H2O 200, CaCl2·2H2O 40, K2HPO4 500, Na2EDTA 80, FeSO4·7H2O 10, and trace elements solution 1 mL L−1. The trace elements solution consisted of: ZnSO4·7H2O 222 mg L−1, H3BO3 2, 860 mg L−1, MnSO4·7H2O 2, 500 mg L−1, CuSO4·5H2O 79 mg L−1, and NaMoO4·2H2O 21 mg L−1. The culture system was 30 m3 open raceway ponds with an
X = 0.61 × OD560
(R2 = 0.99)
(1)
Each sample of 5 mL microalgal suspension was used to measure its OD. Samples were diluted for 10 times to give an absorbance in the range of 0.1–1.0. The optical density measurements were periodically 2
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2.3.5. Measurement of nutrients The samples of algal suspensions for analysis were first centrifuged at 4, 800 × g for 10 min and then the supernatants were collected and stored at 4 °C for further use. They were diluted for 20 times and analyzed for TN, and TP following the Hach DR 5000 Spectrophotometer Manual (Hach, 2008). The supernatants were filtrated using 0.45-μm membrane filters (Millipore Corporation, USA) and diluted for 5 times for analysis. The diluted solution of 0.5 mL was injected into a Multi N/ C3100 Analyzer (Analytik Jena AG, Jena, Germany) for the analysis of the dissolved organic carbon (DOC).
checked by gravimetry. Biomass productivity P (g m−2 d−1) was calculated according to the following equation:
P=
(X2 − X1 + Xh ) × V (t2 − t1) × A −1
(2) −1
where X1 (g L ) and X2 (g L ) are the microalgal cell concentrations at culture time t1 (day) and t2 (day), respectively. Xh is the dry weight of the harvested biomass between culture time t1and t2. V (m3) and A (m2) are cultivation volume and irradiance area, respectively.
2.3.6. Phycocyanin analysis A sample of A. platensis (60 mL) was filtered using the 37-μm opening size sieves, washed with distilled water for three times and the pellets were resuspended in 6 mL of 0.125 mol L−1 sodium phosphate salt solution containing 4 mmol L−1 NaN3, 2 mmol L−1 mercaptoethanol and 2 mmol L−1 EDTA to extract the phycocyanin. The mixture was disrupted using a 600 W ultrasonic cell disintegrator (GA92-IID, Wuxi Shangjia Biotechnology Co., Ltd, Jiangsu Province, China) for 30 s with 5 s intervals for a duration of 10 min followed by centrifugation at 4800 × g for 5 min. The phycocyanin concentration in the supernatants was determined spectrophotometrically by measuring the absorbance at 280, 620, and 652 nm, respectively. The phycocyanin concentration (PCC, g L–1) of A. platensis was calculated using OD according to the following equation (Bennett and Bogorad, 1973):
2.3.2. Measurement of photosynthesis The maximum quantum yield of PSΠ (Fv/Fm), a key parameter indicating photosynthetic performance, was measured using a pulse-amplitude-modulation fluorometer (Heinz Walz Gmbh, Effeltrich, Germany). Each sample of 3 mL microalgal suspension was acclimatized in the dark for 15 min prior to the measurements of the light response curves of photosynthesis. Light response curves were developed by using WinControl software v.3.2. The measurements were carried out in 4-mL cuvettes and the microalgal suspension was mixed during measurements using a magnetic stirrer. Fv/Fm was defined as:
Fv / Fm = (Fm − F0)/Fm
(3)
where F0 and Fm are the initial and maximum chlorophyll fluorescence, respectively. Fv represents the variation in chlorophyll fluorescence between Fm and F0.
PCC =
OD620 − 0.474 × OD652 5.34
(4)
2.3.3. DNA amplification, and high throughput DNA sequencing Samples were collected from the outdoor raceway ponds on day 1, 11, 21, and 31 for the treatment and control, respectively. The microbial genomic DNA was extracted by using a PowerSoil DNA extraction kit (MoBio Laboratories, CA, USA) according to the manufacturer’s recommendations and PCR amplification of the 16S rRNA (V4 region) was carried out. The primers of 515F (5′-GTGCCAGCMGCCGCGG TAA-3′) and 806R (5′-GGACTACHVGGGT-WTCTAAT-3′) were used to target the microbial and archaeal 16S rRNA genes. The PCR system was as follows, within 25 μL reaction system, 10 μL supermix, 0.25 μL of each primer, 1 μL template DNA, and 13.75 μL sterile MilliQ H2O. The PCR program included the steps as bellow: initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, elongation at 72 °C for 30 s, and final extension at 72 °C for 5 min. PCR products were measured by 1% (w/v) agarose-gel electrophoresis. Amplification reactions were done in triplicate, and PCR reaction products were pooled prior to sequencing by using an Illumina MiSeq platform HiSeq 2500 system (Illumina, San Diego, CA, USA) at the Novogene Biotechnology Co., Ltd (Beijing, China). The sequencing was performed, raw reads were obtained, and low-quality fragments were filtered using Mothur as described in previous studies (Kozich et al., 2013). The pyrosequencing procedure, statistical and bioinformatics analysis were conducted.
where OD620 and OD652 are the absorbance of the phycocyanin extracts at 620, and 652 nm, respectively. The phycocyanin purity ratio (PPR) was defined as follows (Walter et al., 2011):
2.3.4. Cell count To calculate the abundance of the microzooplankton, a hemocytometer was used to count the microzooplankton with a microscope (Leica DM 1000, Leica Microsystems (Shanghai) Trading Co., Ltd., China) directly. To enumerate microorganism (except microzooplankton and A. platensis) cell count of the samples, 25.0 mL of each microalgal samples was resuspended in 225 mL sterile saline solution (0.9%). And then they were blended evenly, subsequently diluted up to 10−4 using the sterile saline solution and blended evenly again. At last 1 mL of each diluted samples were plated on the nutrient agar plate, which was consisted of (weight/volume) beef extract (0.3%), peptone (0.5%), sodium chloride (0.5%), and agar (0.2%). The microorganisms were cultured at 37 °C for 48 h, subsequently counted.
3.1. Effect of microfiltered culture medium on cell growth
PPR =
OD620 OD280
(5)
where OD280 is the absorbance of the phycocyanin extracts at 280 nm. The phycocyanin content (PCT) was defined as dry weight ratio of extracted phycocyanin to biomass by using the equation according to Sharma et al. (2014):
PCT =
PCC × 100% X
(6)
where X is the biomass concentration, PCC is the phycocyanin concentration. 2.4. Data analysis In this work, all runs were carried out at random. All trials were performed in triplicate. Analysis of variance (ANOVA) of the data was carried out as described in a previous study (Zheng et al., 2012). 3. Results and discussion
Outdoor raceway ponds are the most common cultivation system for Spirulina in China. The main obstacle to the cultivation system of Spirulina in large-scale outdoor raceway ponds is contamination, which is caused by microalgal contaminants from air, or land during cultivation process, or harvesting process or medium reuse process. The effects of microalgal contaminants on microalgal growth were constraining the growth of microalgae, causing sudden and massive death of microalgal cells, and competing with each other for nutrients and spaces (Wang et al., 2013b). Microalgal contaminants in the medium could be removed by using microfiltration membranes (Ma et al., 2014). The effect of microfiltered culture medium on microalgal growth is shown in 3
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Table 1 The growth performance of A. platensis during continuous cultivation in large-scale outdoor raceway ponds. Experiment
Time (day)
Biomass concentration (g L−1)
P (g m−2 d−1)
TN (mg L−1)
TP (mg L−1)
DOC (mg L−1)
Control
1 11 21 31
0.21 0.62 0.57 0.45
± ± ± ±
0.00 0.01 0.03 0.02
5±0 34 ± 2 32 ± 1 25 ± 2
62.5 56.4 53.9 25.1
± ± ± ±
0.3 0.5 0.2 0.3
32.8 29.3 30.2 16.7
± ± ± ±
0.0 0.1 0.3 0.5
28.6 ± 0.0 91.7 ± 0.3 123.2 ± 0.3 36.5 ± 0.2
Treatment
1 11 21 31
0.22 0.64 0.59 0.57
± ± ± ±
0.00 0.03 0.02 0.01
6±0 36 ± 2 33 ± 1 32 ± 0
62.0 55.3 57.8 20.6
± ± ± ±
0.0 0.1 0.4 0.2
31.9 30.4 29.7 14.2
± ± ± ±
0.2 0.2 0.4 0.2
29.7 ± 0.1 96.6 ± 0.3 106.5 ± 0.2 58.3 ± 0.1
Data are shown as their replicate mean ± standard deviation. The same below.
increasing culture time for both the treatment and control during the initial 4 days. After that, at the harvesting days both the treatment and control had relative lower Fv/Fm than those of the next day, respectively. There were no significant differences (P > 0.05) in the Fv/Fm of the treatment and control during the initial 20 days. After that, the Fv/ Fm of the treatment and control had significant differences (P < 0.05). The Fv/Fm of the control decreased significantly (P < 0.05) at the late days of cultivation. The results indicated that the culture medium for reuse without microfiltration inhibited photosynthesis of A. platensis. The possible reason was that algicidal products from biological pollutants in the culture medium inhibited microalgal photosynthesis (Lam et al., 2018). The results also indicated that microfiltered culture medium was benefit to photosynthesis of A. platensis. Nutrients and pH of the culture were shifted by microfiltration to remove metabolites of algal pieces, and biological pollutants. Fv/Fm of the microalgae was affected by the factors such as nutrients, temperature, light, pH, and algal species (Zheng et al., 2018, 2019; Yao et al., 2016). Fv/Fm of the microalga of the treatment reached peak values of 0.744 on day 8 while it did so of 0.739 on day 12 for the control. During the initial 5 days, biomass concentration of the treatment increased along with increasing culture time (Fig. 1). There were no significant differences (P > 0.05) in biomass concentration of the treatment and control during the initial 20 days. After that, biomass concentration of the control decreased significantly (P < 0.05). Similar results were reported by Yuan et al. (2011). However, biomass concentration of the treatment decreased slightly. The highest biomass concentration and P of the treatment were 0.65 g L−1 and 37 g m−2 d−1, respectively, on day 5. The highest biomass concentration and P of the control were 0.63 g L−1 and 35 g m−2 d−1, respectively, on day 5. During the investigation, both TN and TP of the treatment and control decreased with increasing culture time. On day 31, biomass concentration and P of the treatment were 1.27 times and 1.28 times that of the control, respectively. The results showed that the microalgal growth of the control was inhibited compared with the treatment. The possible reason is that competition for available nutrients between alga and biological pollutants inhibited microalgal growth (Lam et al., 2018). Furthermore, some biological pollutants such as microzooplanktons, which could be removed by microfiltration, consumed algal cells and thus their presence inhibited microalgal growth. The effect of microfiltered culture medium on pH was investigated. Fig. 2 shows that pH values increased sharply during the initial 5 days for the treatment and control, respectively. Similar results were obtained by a previous study (Zhou et al., 2017). Possible reasons for the pH values increase may have been the consumption of nitrate (Bedekar et al., 2010), the transport of hydroxide ions to the outside of the cell through a reaction catalyzed by the enzyme carbonic anhydrase during the conversion of bicarbonate ions inside the cell to provide CO2 for the photosynthetic reaction and the activity of ribulose 1, 5-bisphosphate carboxylase which was significantly dependent on pH, increasing at higher pH. And ribulose 1, 5-bisphosphate carboxylase was present in the photosynthetic apparatus of the microalga, where the H+ ions were
Fig. 1. Photosynthetic performance and biomass accumulation of A. platensis during continuous cultivation in large-scale outdoor raceway ponds (Fv/Fm of the control (▲), Fv/Fm of the treatment (●); Biomass concentration of the control ( ), Biomass concentration of the treatment ( )).
Fig. 2. pH and exopolysaccharide variations of A. platensis during continuous cultivation in large-scale outdoor raceway ponds (pH of the control (▲), pH of the treatment (●); Exopolysaccharide concentration of the control ( ), Exopolysaccharide concentration of the treatment( )).
Table 1 and Figs. 1 and 2. The culture cultivated in the reused culture medium without microfiltration was used as control. Filtrating the medium by using microfiltration membranes could increase production cost of the continuous cultivation of Spirulina because of energy consumption, labor and equipment cost. Based on cost-benefit perspective, the culture medium for reuse during the whole cultivation process was only filtrated for one time on day 21. Fv/Fm from the treatment and control as a function of culture time was investigated. Fig. 1 shows that Fv/Fm increased along with 4
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3.2. Effect of microfiltered culture medium on microbiological contamination
sequestered to the inside of the tylacoid membrane and these light-induced energy fluxes resulted in an increase in pH values and activating the rubisco enzyme (Francisco et al., 2010). After that, pH values increased slightly for the treatment and control. The possible reason is that the supplement of sodium bicarbonate in the crude natural soda alleviated pH increase. Furthermore, pH of the culture medium was stabilized by exopolysaccharides (De Philippis et al., 2001), which were released by A. platensis (Fig. 2). pH values decreased significantly (P < 0.05) after day 20 for the control. It might be that microalgal contaminants of the control increased with increasing culture time and utilize exopolysaccharide to metabolize acidic substances leading to the pH drop. A lot of cyanobacteria were characterized by the presence of polysaccharidic outermost investments (De Philippis et al., 2001). Three kinds of investments including sheaths, capsules, and slimes were found in cyanobacteria. Most exopolysaccharides, which were released into the culture medium as water-soluble polymer, were in the existence of capsules and/or slimes (De Philippis and Vincenzini, 1998). And sheaths only accounted for a small fraction of the cell dry weight. Exopolysaccharides could trap metal ions, alter the rheological behaviour of water, and stabilize the flow properties of the culture medium under drastic variation of temperature, ionic strength and pH (De Philippis et al., 2001). Thus exopolysaccharides produced from microalga would affect itself growth. The effect of microfiltration of the reused culture medium on exopolysaccharide concentration was investigated. Exopolysaccharide concentrations increased from 0.24 mg L−1 to 2.31 mg L−1 for the treatment and from 0.25 mg L−1 to 2.26 mg L−1 for the control during the initial 20 days, respectively (Fig. 2). The exopolysaccharide concentrations for both the treatment and control were decreased greatly after that. DOC of the two cultures, which was caused partially by exopolysaccharides, reached their peaks before they decreased with increasing culture time (Table 1). The peaks of the DOC in the treatment and control were 106.5 mg L−1, and 123.2 mg L−1, respectively, on day 21. DOC decreased at the late days of cultivation with decreasing exopolysaccharide concentration. It may be that exopolysaccharides were composed of the hexoses glucose, galactose, mannose, pentoses ribose, arabinose, etc (De Philippis et al., 2001). Most of them could be used as substrates for microbial growth. Thus exopolysaccharide concentration decreased at the late days of cultivation with increasing the cell density of bacteria and microzooplankton (Table 2). The exopolysaccharide concentration of the treatment had a sharp decline on day 21. This is because that some exopolysaccharides in the culture medium for reuse were removed by microfiltration on day 20 causing the decrease of exopolysaccharide concentration. It indicated that microfiltration had significant effect (P < 0.05) on exopolysaccharide concentration.
To overcome the challenges of microbiological contamination, researchers have been searching for feasible approaches to control them. Previous studies preferred filtrating the culture medium of microalgae to eradicate the microbiological contamination to adding drugs to annihilate the microbiological contamination (Lam et al., 2018; Wang et al., 2013b). Microfiltered culture medium had significant effect (P < 0.05) on the amount of bacteria and microzooplanktons (Table 2). For both the treatment and control, the amount of bacteria and microzooplankton all increased with increasing culture time. A massive outbreak of bacteria and microzooplankton did not occur in this study. The cultures like Spirulina were a limited number of microalgae, which grew successfully in large-scale outdoor raceway ponds because of their extreme growth conditions such as very high salinity and high pH (Wang et al., 2013b). In the present study, natural sodium bicarbonate from the nearby alkaline lakes was applied in the medium for A. platensis cultivation. Therefore, A. platensis was grew under saltalkaline stress conditions. However, very high salinity and high pH decreased the growth of bacteria and microzooplanktons thus minimizing the chances of massive outbreak of bacteria and microzooplanktons (Lee, 2001). Furthermore, A. platensis used inorganic matters to synthesize complex molecules through photosynthesis to support its growth and multiplication. Most of bacteria and microzooplanktons needed organic matters to support their growth and multiplication. Therefore, the chances of massive outbreak of bacteria and microzooplanktons was further decreased due to the used medium in this study. Before day 11, no microzooplankton was observed in the cultures of the treatment and control (Table 2). Before day 21, both the amount of bacteria and microzooplanktons were no significant difference (P > 0.05) between the treatment and control. On day 31, the treatment and control had significant difference (P < 0.05) on both the amount of bacteria and microzooplanktons. Bacterial and microzooplankton taxa of the control on day 31 were 9.3 × 105 CFU L−1 and 1.5 × 104 inds. L−1, respectively, which were about 3 and 7 times that of the treatment, respectively. It indicated that microfiltered culture medium could decrease the amount of bacteria and microzooplanktons. Algal suspensions were collected over an One-month period from continuous algae cultivation systems in large-scale outdoor raceway ponds at Ordos Lvfuyuan Biotechnology Corp. Ltd. The algal suspensions were harvested by centrifugation at 9, 600 × g for 15 min. Community DNA was extracted from harvested biomass samples. In total, 24 samples were processed. The V4 region of 16S rRNA genes was amplified and sequenced, generating about 12 million sequenced amplicons. Following filtering steps that removed algae-derived chloroplast and mitochondrial sequences along with extremely rare sequences and other potential sources of error, 2 million microbial reads were used for further analyses. Microbial communities were characterized in the cultivation of A. platensis of the treatment and control. The composition of these microbial communities was compared across all samples using unweighted UniFrac as a distance metric. Algae cultivation of the treatment and control contained microbial communities that were distinct in terms of phylogenetic structure. The microbial community structures of the treatment and control were analyzed. Taxonomic distributions at the phyla level were summarized for the treatment and control samples (Fig. 3). At the phylum level, the ten most abundant phyla were Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia, Spirochaetes, Lentisphaerae, Deinococcus-Thermus, and Protozoa. They could grow under salt-alkaline stress conditions. The microbiological contaminants were zooplankton, bacteria, fungus, other microalgae and virus during microalgal cultivation in open ponds (Lam et al., 2018). The microbiological contaminants in this study only belonged to bacteria and microzooplanktons. At the phylum level,
Table 2 The cell density of bacteria and microzooplanktons. Experiment
Microorganism count
Time (day) 1
Control
Treatment
Bacterial taxa (CFU L−1) Microzooplankton taxa (inds. L−1) Bacterial taxa (CFU L−1) Microzooplankton taxa (inds. L−1)
11 3
21 4
31 5
9.3 × 105
2.0 × 10
2.6 × 10
3.9 × 10
0
0
2.5 × 103
1.5 × 104
1.7 × 103
3.5 × 104
5.1 × 105
2.8 × 105
0
0
3.0 × 103
2.2 × 103
5
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were Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia, Spirochaetes, Lentisphaerae, Deinococcus-Thermus, and Protozoa for both the treatment and control. It showed that new phyla were appeared with increasing culture time. Since the processes of cultures of the treatment and control were done under unsterile conditions in large-scale outdoor raceway ponds, each process, the initial algal inoculum or the harvesting process or the reused medium, was an opportunity for bacteria and other microbes to enter the community and increased species richness and phylogenetic diversity. Beyond those processes, there were numerous environmental differences during cultivation in large-scale outdoor raceway ponds that might affect microbial populations and cause distinct communities to dominate different cultivation systems. Some of these factors would directly influence microbes (e.g., pH, nutrients), while others (e.g., light intensity, oxygen) had impacts on the growth of A. platensis (Chen et al., 2013), which in turn would influence microbial growth. Filtrating culture medium could remove microbes, which consumed nutrients, and oxygen during their growth and their metabolites influencing medium pH. Once light entered medium, microbes as shading light materials limited the amount of available light for A. platensis. In addition to differences in environmental parameters, the continuous cultivation process may affect microbial community composition. In the continuous cultivation system used here, biomass from half of dense A. platensis cultures of the test was harvested and continuous cultures were not inoculated; occasionally, microbes was still in the reused medium. Because culture communities (A. platensis, microbes, and other constituents) were repeatedly reused for continuous cultivation, this cultivation strategy provided additional generations within which communities may have been affected by the conditions of that reused medium and therefore became increasingly distinct from communities grown with different reused medium.
Fig. 3. Microorganism abundance in phylum level. (PB: Proteobacteria; B: Bacteroidetes; F: Firmicutes; A: Actinobacteria; PL: Planctomycetes; V: Verrucomicrobia; S: Spirochaetes; L: Lentisphaerae; D: Deinococcus-Thermus; PT: Protozoa; O: Others.) (C1: control on day 1, C2: control on day 11, C3: control on day 21, C4: control on day 31; T1: treatment on day 1, T2: treatment on day 11, T3: treatment on day 21, T4: treatment on day 31).
Proteobacteria was the most abundant phylum in the community for all the tested samples. Proteobacteria and Bacteroidetes dominated communities from all the tested samples. On day 1, there were two phyla most abundant, Proteobacteria and Bacteroidetes, for the treatment and control accounted for 97%, and 97% of the microbial communities, respectively (Fig. 3). On day 31, the two most abundant phyla for the treatment and control were accounted for 56%, and 46% of the microbial communities, respectively. The results showed that the total abundance of Proteobacteria and Bacteroidetes was decreased with increasing culture time for the treatment and control, respectively. Considering all samples, Proteobacteria became less prevalent as culture time increased, having relative abundances of 56%, 49%, 39%, and 34% in cultivation grown on day 1, 11, 21, and 31 for the treatment, respectively and 57%, 55%, 40%, and 30% in cultivation grown on day 1, 11, 21, and 31 for the control, respectively. Bacteroidetes also decreased in relative abundance as culture time increased, from 41% abundance in the cultivation of the treatment on day 1 to 38% abundance in the cultivation of the treatment on day 11 to 32% abundance in the cultivation of the treatment on day 21 to 22% abundance in the cultivation of the treatment on day 31 and from 40% abundance in the cultivation of the control on day 1 to 33% abundance in the cultivation of the control on day 11 to 25% abundance in the cultivation of the control on day 21 to 16% abundance in the cultivation of the control on day 31 (Fig. 3). Within each cultivation method, microorganisms were ranked by relative abundance. On day 1, the two dominant phyla were Proteobacteria and Bacteroidetes for both the treatment and control (Fig. 3). Proteobacteria and Bacteroidetes previously have been shown to be the most abundant bacteria in algal cultivation system (Fulbright et al., 2018; Simek et al., 2011). On day 31, the ten dominant phyla
3.3. Effect of microfiltered culture medium on phycocyanin production of A. platensis Culture conditions such as culture medium composition, light intensities, temperature, pH, etc. had a significant impact on the microalgae‘s abilities to synthesize phycocyanin (Chen et al., 2013; Yao et al., 2016; Manirafasha et al., 2018). In order to identify the effect of microfiltered culture medium on phycocyanin production, A. platensis was continuously cultured in large-scale outdoor raceway ponds. The PCT was measured on day 1, 11, 21, and 31. The experimental results are presented in Table 3 and Fig. 4. The PCT on day 1 was much lower than that on day 11, 21, and 31 for the treatment and control, respectively. There was a decrease in PCT of the control when the culture time increased from day 11 to 31. The PCT of 10% of the treatment on day 31 was higher than that of 8% of the control. During the investigation, both PCT and protein content of the treatment and control increased before they decreased with increasing culture time. Similar results were obtained by Manirafasha et al. (2016). Phycocyanin was one of proteins found in A. platensis. The results showed that the variation of PCT was highly related to the change of protein content. The carbohydrate
Table 3 The phycocyanin production of A. platensis during continuous cultivation in large-scale outdoor raceway ponds. Experiment
Time (day)
Carbohydrate content (%)
Protein content (%)
PCC (mg L−1)
PCT (%)
PPR
Control
1 11 21 31
18 20 35 42
± ± ± ±
0 1 2 1
41 45 36 31
± ± ± ±
1 1 2 1
10.5 68.2 57.0 40.5
± ± ± ±
0.0 0.5 0.2 0.8
5±0 12 ± 1 11 ± 0 8±1
0.654 0.713 0.722 0.709
± ± ± ±
0.005 0.009 0.012 0.007
Treatment
1 11 21 31
19 21 32 35
± ± ± ±
0 1 1 2
41 49 43 34
± ± ± ±
0 3 2 1
13.2 76.8 64.9 68.4
± ± ± ±
0.4 0.6 0.7 0.3
6±0 14 ± 1 12 ± 0 10 ± 0
0.660 0.717 0.713 0.720
± ± ± ±
0.004 0.008 0.007 0.003
6
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Strategies to control biological contaminants during microalgal cultivation in open ponds. Bioresourc. Technol. 252, 180–187. Lebleu, N., Roques, C., Aimar, P., Causserand, C., 2009. Role of the cell-wall structure in the retention of bacteria by microfiltration membranes. J. Membrane Sci. 326, 178–185. Lee, Y.K., 2001. Microalgal mass culture systems and methods: their limitation and potential. J. Appl. Phycol. 13 (4), 307–315. Liu, Q., Huang, Y., Zhang, R., Cai, T., Cai, Y., 2016. Medical application of Spirulina platensis derived C-phycocyanin. Evid. Based Complement Alternat. Med. 2016, 1–14. Logan, C., Roanld, S., 2011. Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnol. Adv. 29, 686–702. Ma, H.Y., Hsiao, B., Chu, B., 2014. Functionalized electrospun nanofibrous microfiltration membranes for removal of bacteria and viruses. J. Membrane Sci. 452, 446–452. Manirafasha, E., Ndikubwimana, T., Zeng, X., Lu, Y., Jing, K., 2016. 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Zheng, H.L., Gao, Z., Yin, F.W., Ji, X.J., Huang, H., 2012. Effect of CO2 supply conditions
Fig. 4. Phycocyanin production of A. platensis during continuous cultivation in large-scale outdoor raceway ponds (C: control, T: treatment).
content of the treatment and control all increased along with increasing culture time. To a certain extent, there was a negative correlation between protein content and carbohydrate content. The PCC increased significantly (P < 0.05) during the initial 5 days for the treatment and control, respectively, and decreased slightly during the last 10 days for the control (Table 3 and Fig. 4). The PCC and PPR of the control all increased before they decreased along with increasing culture time. The PCC and PPR on day 1 was much lower than those on day 11, 21, and 31 for the treatment, respectively. The PCC and PPR of the treatment had little variation at the late 20 days, respectively. There was much difference in PCC and PPR on day 31 between the treatment and control. Especially, the highest PCC was 68.2 mg L−1 with a PPR of 0.713 for the control, while the highest PCC was 76.8 mg L−1 with a PPR of 0.717 for the treatment. The results herein presented attest that the hypothesis might be a reality. The PCT and PCC could be enhanced through filtrating the culture medium by using microfiltration membranes prior to reuse.
4. Conclusions A. platensis was able to produce phycocyanin efficiently in the existence of microbiological contamination in large-scale outdoor raceway ponds. Our study is the first report to identify the major harmful microbial species in commercial Spirulina farms. Ten phyla of microorganisms belonging to bacteria and microzooplanktons were identified. A strategy was established to alleviate microbiological contamination by using microfiltration membranes during continuous cultivation of A. platensis. Considering the environmental benefits and product quality, it is a promising way of using microfiltration, which could efficiently remove the microbiological contaminants, for the treatment of the culture medium for reuse subsequently.
Acknowledgements This work was supported in part by grants from the National Natural Science Foundation of China (Grant No. 21767017), and the Science and Technology Project of Jiangxi Provincial Department of Science and Technology, China (Grant Nos. 20171BBF60024 and 20181BBF60026), and the Innovation and Entrepreneurship Development Fund of ‘‘Thousand talents program” Talent of Jiangxi Province, China (Grant No. 1001-02102082).
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