Fengzheng Gao, Guangxin Feng, Wei Guo, Mingyong Zeng*
College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, Shandong Province, 266003, China
* Corresponding author.
Mingyong Zeng, Tel.: +86-532-8203-2783; E-mail: email@example.com.
Received 01-12-2017; received in revised form 24-04-2018; accepted 25-04-2018
Abstract Inorganic polyphosphate bodies (PPB) have been linked to a variety of functions recently. To explore the PPB in the cells of Synechococcus sp. PCC 7002, transmission electron microscopy, dynamic light scattering, and energy dispersive X-ray (EDX) analysis were utilized to observe and analyze the PPB. PPB from Synechococcus sp. PCC 7002 were nanosized particles ranged from 40 nanometers to approximately 100 nanometers in diameter and mainly located around the periphery of the cells. The yield of PPB increased with biomass and reached maximum at early stationary phase. EDX analysis showed that PPB contained many mineral elements such as iron, calcium, and magnesium. As cyanobacteria with fast growth rate and substantial biomass, Synechococcus sp. PCC 7002 is a potential microbial cell factory for PPB production.
Keywords: polyphosphate bodies; Synechococcus sp. PCC 7002; mineral elements; microbial cell factory
Microalgae are considered one of the most promising feed stocks for sustainable production of food, feed, chemical, material, and fuel (Draaisma R.B et al., 2013). Potential high-value products from microalgae include food supplement, omega fatty acid, pigment, specialty oil, antioxidant, functional protein, and specific polysaccharide for application as valuable ingredients in food, aquatic feed, and cosmetics (Wijffels R.H et al., 2010). Microalgae have been regarded as a promising and powerful cell factory for their significant role in global sustainability initiatives (León-Bañares R. et al., 2004; Fu W et al., 2016). There is an increasing interest in the development of bioactive products from microalgae. More and more attention has been paid to the variety of functions and high value of microalgae (Ruiz J et al., 2016).
Polyphosphate bodies (also known as volutin granules) are mineralized structures present in most of the cells, as well as microalgae cells, and are mainly composed of long-chain PolyP (Segawa S et al., 2011; Sakatani A et al., 2013; et al., 2015). Biomineralized nanostructural particles possess unique functional properties that make them suitable for nutrition intervention programs. Mineral nanoparticles can be absorbed by intestinal epithelial cells in form of whole particles with high absorption and utilization efficiency (Wu H et al., 2014).
Cells of Synechococcus were reported having nanosized PPB, such as Anacystis nidulan (PPB size approximately 200 nm) (Lawry N.H et al., 1979), thermophilic Synechococcus sp. from microbial mats (PPB size from approximately 100 nm to 300 nm) (Gomez-Garcia M.R et al., 2013) and Synechococcus elongatus PCC 7942 (PPB size above 300 nm) (Murata K et al., 2016). However, PPB from cells of Synechococcus sp. PCC 7002 was not reported yet. It is necessary and important to observe and analyze the PPB from one of the most typical cyanobacteria, Synechococcus sp. PCC 7002.
The ability of cyanobacteria to utilize sunlight for capturing CO2 makes them powerful cell factories (Pinto F et al., 2015). Synechococcus sp. PCC 7002 with known gene sequences and rapidly divisive speed has long been a model transgenic organism alongside with Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 (Hendry J.I et al., 2016). It is a good platform for gene manipulation with fast growth rate and naturally transformable capability. Synechococcus sp. PCC 7002 was utilized to produce many bioactive products by transgenic engineering (Pérez A.A et al., 2016; Dong X.W et al., 2016; Mahadev S.R et al., 2013). These make it an ideal candidate for the production of biofuels and commercially valuable secondary metabolites. Polyphosphate kinase (ppk) gene was found to have an important link with the production of PPB in cells (Rao N.N et al., 2009). Chen (2013) reported that the overexpression of the ppk gene in Synechococcus elongatus PCC 7942 improved the accumulation of PPB in cells and then improved the ability of heavy mental tolerance. As genetic manipulation model microalgae, Synechococcus sp. PCC 7002 has the potential to overexpress ppk gene by foreign recombinant plasmid import and expression.
From the above, we assume that Synechococcus sp. PCC 7002 is an excellent cell factory for PPB production.
2. Materials and methods
The Synechococcus sp. PCC 7002 strain used in this study was kindly provided by Prof. Jindong ZHAO from the Institute of Hydrobiology, Chinese Academy of Sciences, and was cultured in artificial seawater with A medium (Bernstein et al., 2014) at 32 °C with 100 µmol/m2·s light intensity.
2.2 Observation of Synechococcus sp. PCC 7002 cells
Transmission electron microscopy (TEM) is usually used to observe the PPB in cells. Synechococcus sp. PCC 7002 was cultivated in 250 mL flasks with 100 mL medium until the OD750 value of the culture reached around 2. An amount of 1.5 mL cells was harvested by centrifuge at 9500 g for 1 min at room temperature. The cells were cleaned with fresh medium and normal saline and then were centrifuged again. The clean cells were fixated with 2.5% glutaraldehyde for 24 h at room temperature and then were stored at 4 °C. After fixation, the cells were centrifuged at 9500 g for 10 min and the supernatant was discarded. The ultrathin sections were prepared and observed by TEM.
2.3 PPB extraction and measurement
PolyP has characteristic fluorescence at 550 nm with DAPI without prior purification (Kulakova A.N. et al., 2011). The PPB were extracted according to the method from Martin (Martin P. et al., 2013). Cells were harvested from 1 mL aliquots of culture medium at the same time every day at 9000 g for 1 min by centrifuge. The pellets were re-suspended with 1 mL HEPES buffer (20 mM HEPES pH 7.0, 150 mM KCl) and were boiled with 100 °C water for 10 min. After boiling and cooling to the room temperature, the samples were digested by 200 μg/mL proteinase K at 37 °C for 30 min. The liquid supernatant was obtained by centrifugation at 16 000 g for 10 min at room temperature. PPB was measured by HITACHI F-4600 Fluorescence Spectrofluorometer according to the method from Martin (Martin P. et al., 2013).
2.4 Particle size analysis
The extracted PPB was filtered through 0.22-μm cellulose acetate filters before analysis. Dynamic light scattering (DLS) was arranged to measure the sizes of PPB by number. The samples were diluted properly if the result of pretest did not match the analysis requirement.
2.5 PPB observation and EDX analysis
The extracted PPB samples (DAPI fluorescence around 100 kcps) were diluted 1000 times with Milli-Q water and fixed on the copper grids and observed by TEM. For elementary composition measurement, the samples were analyzed by EDX.
3. Results and discussion
3.1 PPB observation
PPB can be observed as black spots clearly under TEM. As shown in Fig.1, the PPB from Synechococcus sp. PCC 7002 distributed mainly around the periphery of the cells. However, other microalgae (Lawry N.H. et al., 2014; Gomez-Garcia M.R. et al., 2013; Murata K. et al., 2016) mentioned above, the PPB mainly distributed in the center of the cells. Synechococcus sp. PCC 7002 is a small microalga with diameter range from 0.5 to 2 micrometers. The sizes of PPB from Synechococcus sp. PCC 7002 are nanosized and smaller than that from other Synechococcus species. Most of them are below 100 nm in diameter. Generally, the absorption efficiency will increase with decrease of the particle size. The better absorption efficiency can be expected with smaller PPB. Natural nanosized products have the advantage in the field of food and medicine.
Fig.1 TEM images of PPB in the cells of Synechococcus sp. PCC 7002 a: Image of cells; b: Further enlarged image of cell.
Microalgae are recognized as nutritious food resources with a variety of functions and rich in mineral elements. Some previous studies reported the utilization of microalgae in the field of heavy metal removal (Yang S. et al., 2017). Eatable microalgae, such as Spirulina and Chlorella, are functional mineral nutrition food resources. Up to now, no toxin was found to be produced from Synechococcus sp. PCC 7002. That makes it possible to develop Synechococcus sp. PCC 7002 to a new food resource. The whole cell of Synechococcus sp. PCC 7002 as well as productions like PPB can be used in functional food industry.
3.2 PPB measurement during different growth phases
As shown in Fig.2, DAPI fluorescence of PPB increased with biomass increase. Synechococcus sp. PCC 7002 was cultivated in the flasks without aeration; the final biomass was not very high. The yield of PPB was the highest on day 7 at the early stationary phase. After then, it dropped significantly. The content of phosphorus in the medium is an important factor for the accumulation of PPB in the cells. PPB was utilized by the cells at the late stationary phase when the phosphorus was limited. PPB accumulation is a strategy for cells to survive from limited phosphorus conditions. It also provided us an idea to improve the yield of PPB by using phosphorus rich medium. In addition, much more biomass can be harvested for PPB extraction if photobioreactors with CO2 supplementation are arranged for the cultivation.
Fig.2 Growth curve of Synechococcus sp. PCC 7002 and yield changes of PolyP
PPB accumulated during the growth of Synechococcus sp. PCC 7002 and the maximum appeared at the early stationary phase, and the next steps for PPB production should be photobioreactors cultivation and medium optimization; both are practical methods to improve the yield of PPB.
3.3 Particle size analysis
The extracted PPB was dissolved with Milli-Q water and analyzed by DLS. As we can see from Fig.3, the sizes of most PPB ranged from tens to one hundred nanometers. PPB with the size of 50 nm was the most with a percent of nearly 25%. A small amount of PPB was above one hundred nanometers. The size of PPB is not stationary during the growth. It changed with the growth of cells and affected by the cultivation conditions. As Seki reported, the average size of polyphosphate bodies from Synechococcus elongatus PCC 7942 increased gradually without signiﬁcant change in their number and distribution during the dark period (Seki Y. et al., 2014). However, during the light period, the number of polyphosphate bodies increased, while the size of each polyphosphate body decreased until the end of the light period (Seki Y. et al., 2014).
It will be interesting if we can produce PPB with expected size by controlling the cultivation conditions. Nanoparticles are excellent nutritional functional substances, especially for those extracted from marine organisms. Unlike the artificial nanoparticles, PPB from Synechococcus sp. PCC 7002 was a natural product and can be mass-produced via large-scale cultivation.
Fig.3 Size arrange of PPB from Synechococcus sp. PCC 7002
3.4 Elemental analysis
Samples analyzed by DLS were fixed and observed under TEM. Clear black spots of different sizes can be observed in Fig.4. PPB was maintained in a good nano-shape after 10-min boring extraction procedure. This suggested that they have high thermal stability.
Fig.4 TEM images of the extracted PPB a: Image of PPB; b: Further enlarged image of PPB.
To analyze the elemental composition, EDX was utilized during the TEM observation. As Figure 5 shows, PPB contains several elements. Many carbon and cuprum were recognized from the spectrogram because the PPB were fixed on copper grids with carbon support membrane. The culture medium for Synechococcus sp. PCC 7002 was seawater medium with 18 g/L NaCl, high concentrations of sodium and chlorine were observed from the spectrum. PolyP is a linear polymer composed of three to several hundred phosphate groups that is likely produced in the cells of all organisms. As a characteristic element of PPB, phosphorus content was high according to the height of the peak on the spectrum.
Fig.5 EDX analysis of PPB
Mineral elements, iron, calcium, and magnesium were also found in PPB. This result was similar to that of Vossbrinck (Vossbrinck C.R. et al., 1994) and Jensen (Jensen T.E. et al., 1993). The ability of storing mental elements in PPB helps microalgae survive at hostile conditions of high concentration of mental elements. The heavy metal tolerance ability of the cells was improved significantly with more PPB, as PPB can store surplus metal element. As natural mineralized nanoparticles, PPB has a potential application in the field of mineral nutrition reinforcement.
Mineral deficiency is one of the most prominent global health risks. The efficiency of absorption and utilization of chemical synthesis of mineral nutrient fortifier is low. It is always a hot topic to find the new nutrition fortifiers. The producing of PPB from microalgae provides a new idea to produce natural functional particles using light and carbon dioxide. Except for the elements shown on the energy spectrum, there must be other elements with low content so that they cannot be detected by instruments. It is important to supply several mineral elements at the same time during the strengthening process, for people are likely to face with multiple nutrient elements deficiency problems.
To improve the value of the microalgae, new functional substances from microalgae should be developed and utilized (Ruiz J. et al., 2016). PPB is rich in mineral elements, especially iron and calcium, which makes it possible to be developed as a mineral nutrient fortifier. The concentration of the mineral elements can be improved in the medium to produce PPB with high concentration of a mineral. It provided us a promising way to produce new nanosized particles from microalgae.
We observed and analyzed the PPB in the cells of Synechococcus sp. PCC 7002 using TEM, DLS, and EDX. PPB from Synechococcus sp. PCC 7002 is nanosized particles rich in mineral elements and mainly distributed around the periphery of the cells. It will be an interesting research topic to produce PPB by microalgae. Synechococcus sp. PCC 7002 is a potential microbial cell factory for polyphosphate body production.
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