2. Materials and Methods
2.1 Media and Chemicals
Minimalist salt medium (MSM) used for producing P. putida F1 was prepared in our lab. MSM solution lacking carbon source substrate was made by dissolving chemicals below to a solution of final concentrations as: NaNO3 (1 g L-1), KH2PO4 (1 g L-1), K2HPO4 (1 g L-1), MgSO4 (0.2 g L-1), KCl (0.7 g L-1), NaCl (0.3 g L-1), CaCl2 (0.02 g L-1), EDTA (2 mg L-1), FeCl2·4H2O (1.5 mg L-1), H3BO3 (0.06 mg L-1), MnCl2·4H2O (0.1 mg L-1), CoCl2·2H2O (0.12 mg L-1), ZnCl2 (0.07 mg L-1), NiCl2·6H2O (0.025 mg L-1), CuSO4·5H2O (0.02 mg L-1) and NaMoO4·2H2O (0.025 mg L-1). For MSM solid plate, extra agar (15 g L-1) was added along with the above chemicals. For concentrated MSM, the concentration of the chemicals dissolved was doubled as MSM solution. Luria-Bertani (LB) media for producing P. putida F1 culture to anchor biomass onto cellulosic carbon fibers consisted of NaCl (10 g L-1), peptone (10 g L-1), and yeast extract (5 g L-1). Toluene was purchased from Fisher Scientific (Waltham, MA). All the other chemicals were purchased either from Fisher Scientific or Sigma-Aldrich (St. Louis, MO).
2.2 Strain and Cultivation
Pseudomonas putida (Trevisan) Migula (ATCCⓇ 700007TM) was selected as a model bacterium in this study due to its ability of consuming toluene (Zylstra et al., 1988). The strain was preserved in a -70 °C refrigerator. To reactivate P. putida F1, a 2 L glass bottle containing 200 mL of MSM was inoculated with 1 mL of preserved strain and capped with a Teflon-lined silicon septum having a glass bulb with a small hole penetrating through. Toluene vapor was supplied continuously through the small hole on the glass bulb as the sole energy and carbon source (Claus & Walker, 1964). The bottle was then incubated in a shaker having 150 rpm and 30 ℃ under aseptic conditions for 24 hours. For further use, the activated P. putida F1 was inoculated onto an MSM solid plate and stored in a 4 °C refrigerator.
2.3 Preparation of Carbonized Cellulosic Fiber (CCF)
Woven gauze sponges (10 × 10 cm, 100 % cotton) were selected as the cellulosic material for carbonization as it has uniform structures and high purity and requires no pretreatments. It was later carbonized in a high-temperature tubular furnace (Sentro Tech., Model STT-1200-3.5-12, Cleveland, OH). A typical cellulosic carbon fiber (CCF) production process was described in our previous work (Wang et al., 2021). In summary, gauze sponges were placed in the high-temperature tubular furnace and then heated up to 250 ℃ for 3 hours with argon fed to the furnace at a flow rate of 500 mL/min. The temperature of the furnace was then increased to 650 ℃ for 4 hours and the feed was changed to a hydrogen and argon gas mixture using flow rates of 150 mL/min and 450 mL/min, respectively. Subsequently, the furnace was further heated up to 850 ℃ and was held for 30 min under argon only. The chamber was finally cooled down to room temperature at the end of the process. The strips of CCF were then removed from the furnace and weighted. The weights were adjusted to 0.17 g by removing excess CCF if needed.
2.4 Analytical Methods
For GC analysis of toluene and CO2, the concentrations of toluene and CO2 were analyzed in an Agilent 6890N Gas Chromatograph equipped with FID and TCD detectors (Agilant.inc.; Santa Clara, CA). A capillary HP-PLOT/Q column (Agilent, Santa Clara, CA) with a length of 30 m and a diameter of 0.530 mm was used for toluene and CO2 determination. For toluene analysis, the temperatures of the oven, injector, and FID detector were maintained at 60 °C, 220 °C, and 260 °C, respectively. For CO2analysis, the temperatures of the oven, injector, and TCD detector were maintained at 60 °C, 220 °C, and 250 °C, respectively.
Scanning electron microscopy (SEM) images of the CCF-supported biofilms were taken with a HITACHI S3500N microscope (Hitachi High Technologies America, Inc.; Schaumberg, Illinois), using 5-10 kV accelerating voltage. Sub-milligram selections of CCF were taken from samples. The sub-samples were then fixed, dried, and dehydrated according to reference (Hazrin-Chong & Manefield, 2012), after which they were coated with 2.5 nm of gold-platinum by the mean of a Cressington 108 Auto sputter coater (Watford, England).
2.5 Preloading P. putida F1 on CCF
MSM media was inoculated with P. putida F1 and incubated overnight. The OD of the culture was then adjusted to 0.5 for later use. For preloading P. putida F1 onto the CCF, five 50 mL centrifuge tubes containing 0.17 g of the CCF were autoclaved and then inoculated with 40 mL of the culture. Then four of the tubes were supplemented with either 2 mM sucrose, 2 mM cystine, 0.4 % (wt/v) citric acid, and 0.5 % (wt/v) NaCl (Chang et al., 2007; Dahlstrom et al., 2018). Finally, the tubes were statically grown (without shaking) for 48 hours at 30 ℃ for biofilm formation.
LB media was inoculated with P. putida F1 and incubated overnight. The OD of the culture was then adjusted to 0.5, 1.0, and 5.0, respectively, for later use. For anchoring P. putida F1 onto the CCF, four 50 mL centrifuge tubes containing 0.17 g of the CCF were autoclaved. Then three of the tubes were inoculated with 40 mL of one of the three LB based cultures. Finally, the tubes were statically grown (without shaking) for 48 hours at 30 ℃ for biofilm formation.
For P. putida F1 biomass density analysis, biomass concentrations in liquid phase were determined by measuring the turbidity of 1 ml of samples of media at 600 nm with a Varian Cary® 50 UV-Vis Spectrophotometer (Varian, Inc.; Lake Forest, CA). The two values were correlated using a regression line that was made with standards ofP. putida F1 during log-phase growth which were diluted to specific turbidity readings and then dried at 90 ℃ in glass tubes overnight and weighed. A linear fit with an R2 of 99.9 % was achieved with triple batches and found a 0.435 optical density correlated to 1 mg ml-1 of dry cell weight between an optical density of 0.2 to 1.
Biomass loading of supported P. putida F1 biofilms was obtained by monitoring dry weight of the samples. After being treated with either the MCM culture or the LB culture, the samples were removed from the centrifuge tubes and washed twice using deionized water. The samples were then placed under 90 ℃ in an oven for overnight drying before being further conditioned at room temperature for 30 min. The dry weight ofP. putida F1 on CCF was calculated based on mass balance using dry weight data of samples before and after preloading.
2.6 Gas-phase P. putida F1 growth in Stationary State
The testing chamber was built using an incubator (Barnstead Lab-Line, Inc.; Melrose Park, Illinois) which allows tubing connections. Toluene vapor was supplied into the testing chamber through the tubing and the waste gas was removed on another side. The testing chamber was also equipped with a humidifier (Great Innovations, LLC.; Miramar, Florida) that was used for adjusting the humidity. To verify the reliability of the testing chamber, two rounds of toluene concentration tests were conducted, and each round lasted for at least five consecutive days. For monitoring the toluene concentration, air samples were collected from the testing chamber and then analyzed via GC-FID/TCD.
For testing of effect of humidity, five different tests were conducted with relative humidifies (RH) of 40, 50, 60, 70, and 80 %. Each test lasted for five consecutive days, and the toluene concentration was set as 60 ppm. Before putting P. putida F1 loaded CCFs the into the testing chamber, each sample was washed with MSM media twice and then left for a short duration until no droplets came from the surface. For each test, six sets of P. putida F1 loaded CCFs were prepared, containing a set of control samples. Each set had three pieces of the well-prepared samples. A set of samples were removed from the testing chamber every day for monitoring the biomass growth.
For evaluation of toxicity of toluene substrate, two different tests were conducted with toluene concentrations of 300 and 1200 ppm. Each test lasted for five consecutive days, and the RH was set at 80 %. Before putting P. putida F1 loaded CCFs into the testing chamber, each sample was washed with MSM media twice and then drained briefly until no droplets came from the surface. For each test, six sets ofP. putida F1 loaded CCFs were prepared, containing a set of control samples. And each set have three pieces of the well-prepared samples. A set of samples was removed from the testing chamber every day for monitoring the biomass growth.
For biofilm stability test, RH and toluene concentration were set as 80% and 60 ppm, respectively. Sample preparation and cultivation protocols were the same as described above unless specified otherwise. For toluene toxicity test, the toluene was only supplied for 6 hours every single day to avoid excess toluene accumulation. For nutrition limitation test, samples were washed by the concentrated MSM media solution for providing the biofilms with more nutrition.
2.7 Long-Term Biodegradation Using Tubular Packed Bed Reactor
For tests with tubular packed bed reactor (TPBR), three pieces P. putida F1 loaded CCFs were produced and then placed into a tubular packed bed reactor. The CCF-biofilm was washed with MSM media twice before being loaded. The TPBR was constructed using 100 ml glass column connected with air flow of 45 ± 5 ml/minute containing toluene contaminated air of 80 ± 5 ppm and RH of 80 % at 30 ℃. Before putting in samples, the TPBR was set for pre-running for 72 hours until the toluene concentration and RH became stable. During 240-hour operation of TPBR, influent and effluent toluene and CO2concentrations were monitored with GC-FID/TCD as described above.