Introduction -- Intracellular polyP accumulation is generally involved in research fields such as environment science and metabolic engineering. PolyP content of microorganisms can be determined by indirect methods, such as biomass and supernatant phosphate concentration determination, or direct methods, such as DAPI-polyP fluorescence measurement and total phosphorous determination after cell lysis [1,2]. In addition to their own advantages and disadvantages, both types of method share a common disadvantage, that is, they are not intuitive. Theoretically, as an intracellular body, it can be visualized intuitively after proper staining, because what we see is what we get. Therefore, to develop a quick polyP staining method is still necessary. The whole procedure only involves four simple steps:

    (1) heated a glass slide,

    (2) smear the sample-dye (toluidine blue) mixture on the preheated glass slide,

    (3) wash the excess dye with water,

    (4) dry the slide with hair dryer.

    Microscopic examination showed that this method can be used to quickly determine whether polyP granules are formed.

    Independent test and feedback -- We verified the feasibility of this method by subjecting it to test by two independent labs led by Prof. Zhonghua Liu (Nanjing Normal University, NNU) and Prof. Liuyan Yang (Nanjing University, NJU). After testing Pseudomonas Putida KT2440 and Citrobacter freundii ATCC 8090 (both are biosafety level 1 species), they told us that this method is simple and sensitive, and they are willing to share this method with researchers in relevant field. Meanwhile, the lab members from NNU pointed out that, in addition to college lab, the clinical laboratory could be the potential audience too, because some pathogens are characterized by intracellular polyP formation. Moreover, both lab members suggested that, as far as intracellular polyP synthesis is concerned, it would be better to perform relative quantification based on these microscopic images, and as such, they can determine the end point of the fermentation process. All these feedbacks are very constructive to our project.

    Introduction -- Despite significant advance in biotechnology and improvement in fermentation control, microorganism contamination remains the major concern in the fermentation process. As a valuable product derived from fermentation, polyP production suffered the same problem.

    Most bacteria cannot grow in the medium where phosphite serves as the phosphorus source. This notion motivates us to test the idea that MRP (engineered E. coli K12 that harbors phosphite dehydrogenase and polyphosphate kinase) can be used to produce polyP under unsterile conditions. We therefore performed serial cultivation experiment using MP (engineered E. coli K12 that overexpresses polyphosphate kinase) as the control. After 8 generations of serial cultivation (12 h per generation), MP that cultured in SMW-P5+ (synthetic municipal wastewater medium with phosphate as the phosphorus source) suffered contamination, as evidenced by the medium color change. For MRP that cultured in SMW-P3+ (synthetic municipal wastewater medium with phosphite as the phosphorus source), the culture stayed pure.

    Independent test and feedback -- We shared this finding with the research team (lead by Prof. Liuyan Yang) in school of the environment in NJU, where they are conducting phosphate removal from wastewater. They helped to scale up the unsterile polyP production using their open membrane reactor. After 96 h of operation, they found no signs of contamination and obtained about 200 mg of pure polyP. Both of us are excited about such output. Meanwhile, they posed questions about the market demand of polyP - more general or just confined in the reagent market. We think this is a really good question which may help us to balance the production of intermediate and final product, if our project is fully developed.

    Introduction -- Incorporation of flame retardant in polymer materials is very important nowadays, because flame retardants are essential in delaying the production and spreading of flames or fires. Among the flame retardants, ammonium polyphosphate had been extensively applied in industries to enhance the fire safety of polymer materials owing to its high phosphorus and nitrogen contents [3]. Its smaller loadings at lower cost and excellent processability make it better than other flame retardants. Most importantly, ammonium polyphosphate is a halogen-free flame retardant and thus it does not generate additional amount of smokes, making it environmentally friendly [4]. Our biological origin polyP is rich in phosphorus either and halogen free. In addition, compared to ammonium polyphosphate with short chain-length polyP (3 to 5-mer), the biological origin polyP is composed of polyP molecules of long chain-length (30 to130-mer). We thus wondered whether the biological origin polyP can play the role of flame retardant as well.

    Independent test and feedback -- To figure out the aforementioned possibility, we designed a unique experiment that independently performed by environment materials lab. We collected MRP cells with intracellular polyP granules and sent them to the college of resources and environmental sciences in Nanjing Agricultural University. Meanwhile, we provided MR (engineered E. coli K12 that harbors phosphite dehydrogenase but cannot synthesize polyP) cells to serve as the control. We hoped they can test our bacteria in extreme conditions - heating to 800°C using their ceramic fiber muffle furnace. Both of us were shocked by the results. After heating, MRP cells converted to biochar whereas MR cells turned to be ashes. This result demonstrated that polyP significantly inhibits the oxidation of carbon under extreme high temperature. Although they were very interested in this phenomenon brought by polyP and believed that new flame retardant still has broad market and development prospect, they also held some questions that mainly focused on the cost of polyP production and the true effects when polyP was used as flame retardant in industries. We believe that these questions can be answered only if we put more effort into the specific research on polyP.

    Introduction -- With the significant progress in biotechnology, we now have various mature media for daily bacteria cultivation. Among them, the most popular one used to culture E. coli is perhaps the LB medium, which is formulated with tryptone (which can be considered as the meat extract), yeast extract, and sodium chloride. However, for biological project aiming to be carried out on terrestrial planets, it should be aware that those media that produced from the secondary producer may not be available at the very initial stage. Under such circumstance, developing one type of natural medium from the primary producer, such as algae, could be a more rational choice. This year in our project, we found that lysate of the blue-green algae can be used to support the growth of E. coli . Based on this finding, we subsequently developed a series of media in different forms using algae as the raw material. Meanwhile, for each form of medium, the storage condition and using method are introduced.

    Independent test and feedback -- To figure out whether the algae lysate can be used to culture other bacteria, we commissioned independent culture test on Pseudomonas Putida KT2440 and Citrobacter freundii ATCC 8090. Results from labs of NJU and NNU both showed that their bacteria can assimilate the lysate for cellular growth. After they heard about the reason why we developed such product, they suggested that, to confirm whether it could be a general option in future application, more tests are needed to be performed, especially on bacterium species that generally adopted as the chassis. In addition, they also hope we can air-dry the lysate and grind it into powder. We benefit greatly from their nice suggestions, which we believe can help us commercialize our research project.