Example 1
Optimization of Growth Medium for Paracoccus sp. when Using Formate as the C1 Compound
[0257]Paracoccus zeaxanthinifaciens ATCC 21588, Paracoccus versutus ATCC 25364, and Paracoccus denitrificans ATCC 13534 were obtained from the American Type Culture Collection (ATCC).
[0258]Strains were tested for the ability to grow aerobically on sodium formate as a sole source of carbon and/or energy using MOPS minimal medium (Teknova, Inc.) with sodium formate as a sole carbon source at 37C. Unlike previous media used to evaluate the formate-dependent growth of Paracoccus, this medium contains defined levels of trace elements molybdenum, boron, copper, zinc, manganese, and other trace metals.
[0259]Growth was conducted in various high-throughput machinery capable of monitoring growth by light scattering at 600 nm, including a Gemini SpectraNax plate reader (Molecular Devices, Inc.), a Tecan M3000 plate reader (Tecan, Inc.), and a BioLector device (m2p-labs, Inc.). For the BioLector, the CO2 gas content in the culture headspace was controllable, as was the humidity.
[0260]Paracoccus zeaxanthinifaciens ATCC 21588 was incapable of growth on formate as a sole carbon or energy source. The other two Paracoccus strains were capable of growth on formate as a sole carbon source, as has been previously reported [Microbiology, 1979, 114(1):1-13, DOI: 10.1099/00221287-114-1-1; Arch Microbiol, 1978, 118(1):21-26, DOI: 10.1007/BF00406069].
[0261]The effect on growth rate of changes in temperature (T, in degrees Celsius), the partial pressure of CO2 in the culture headspace gas (pCO2, in percent by volume), shaking speed (in rpm), and/or the concentrations (in mM) of sodium formate (HCOONa), sodium nitrate (NaNO3), sodium thiosulfate (Na2S2O3), sodium chloride (NaCl), and sodium bicarbonate (NAHCO3) added to the Basal MOPS minimal medium were systematically evaluated. Combinations examined are shown in Table 2. Paracoccus strains are labelled according to their ATCC number. For each measurement, the instrument used (BioLector; SpectraMax; Tecan), plate type (Flower, Flower Plate; 96 transp, 96-well transparent microtiter plate; 96 opaque, 96-well opaque microtiter plate), use of plate lid, use of humidity control and total culture volume in uL is indicated. N/A indicates that a particular experimental condition is not applicable for the instrument used.
TABLE 2 Tested growth conditions for each Paracoccus strain Shaking Humidity ATCC NaHCO3 NaCl NaNO3 Na2S2O3 HCOONa T speed Instrument Plate Lid Control pCO2 Vol 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 20.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 150.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 60.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 60.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 80.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 20.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 20.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 150.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 60.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 60.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 80.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 20.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 10.7 4.3 4.3 40.0 45.7 36.6 1157 BioLector Flower N/A TRUE 5% 1300 13534 75.0 4.3 4.3 5.7 45.7 36.6 1157 BioLector Flower N/A TRUE 5% 1300 13534 10.7 4.3 30.0 5.7 45.7 36.6 1157 BioLector Flower N/A TRUE 5% 1300 25364 42.9 17.1 17.1 0.0 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 13534 0.0 17.1 17.1 22.9 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 13534 42.9 17.1 0.0 22.9 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 25364 101.0 6.1 6.1 11.4 26.7 34.4 937 BioLector Flower N/A TRUE 5% 1300 25364 101.0 6.1 6.1 0.0 26.7 34.4 937 BioLector Flower N/A TRUE 5% 1300 13534 122.4 29.4 14.7 16.3 67.8 34.4 937 BioLector Flower N/A TRUE 5% 1300 13534 122.4 29.4 14.7 0.0 67.8 34.4 937 BioLector Flower N/A TRUE 5% 1300 21588 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 21588 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 21588 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 21588 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE FALSE 1300 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE FALSE 1300
[0262]The particular values for salt, bicarbonate, formate, thiosulfate, or nitrate concentration, as well as temperature, were chosen by implementing a Nelder-Mead simplex optimization algorithm (as described in Chapter 18 of Chemometrics: a textbook ISBN: 0444426604) using the starting simplices with points chosen from the following possibilities: temperature, 34° C. or 37° C.; sodium bicarbonate, 150 mM or 0 mM; sodium chloride, 60 mM or 0 mM; sodium formate, 20 mM or 50 mM; sodium nitrate, 60 mM or 0 mM; sodium thiosulfate, 0 mM or 80 mM; shaking speed, 1200 rpm or 900 rpm. Growth was evaluated for both 25364 and 13534 at each chosen medium condition. For each strain and medium condition, a score indicative of the growth was calculated as the time (in hours) to 50% of the maximum growth attained in the entire experiment minus the time to 5% of the maximum growth. This metric is easy to compute and avoids penalizing conditions with longer lag phases.
[0263]From the scores, new medium conditions were calculated according to the Nelder-Mead simplex algorithm. These medium conditions were tested as well. The growth under the new condition as well as the old ones was used to define the points of a new simplex, and the process repeated.
[0264]After several rounds of the medium optimization process, a satisfactory medium condition, allowing for faster growth than the initially chosen medium conditions, was obtained. In total, 68 different unique medium/strain/temperature/shaking conditions were examined. The fastest growth on formate as a sole carbon source was obtained for ATCC strain 25364. Under the optimal conditions, the medium consisted of 100 mM sodium bicarbonate, 6 mM sodium chloride, 6 mM sodium nitrate, 11 mM sodium thio sulfate, and 26 mM sodium formate in addition to standard MOPS minimal medium components. The optimal growth temperature was 34° C. Under these conditions, ATCC strain 25364 had a growth rate of >0.7 hr−1, corresponding to a doubling time of 0.95 hr.
[0265]ATCC strain 25364 was found capable of growth at rates in excess of 0.4 hr−1 using a simpler medium composition of MOPS minimal medium plus 50 mM sodium formate.
[0266]Large concentrations of thiosulfate were found to give slowed, biphasic growth curves for both strains of Paracoccus tested. In general, moderate concentrations of sodium nitrate showed improved growth. Growth at 34° C. was slightly better than growth at 37° C., and both of these temperatures were significantly better than growth at 30° C.