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.