C ITROBACTER FREUNDII H3 USING D IFFERENT S UPPORTS A S SURFACE
5.3 Material and methods
5.3.3 Batch dark fermentation
Batch cultivations were performed in serum bottle of 100-ml fitted with gas-tight crimp-top rubber septa and flushed with Ar for 15 min and having working volume of 25 ml at a constant temperature of 37oC and shaking at 200 rpm. Cultures were inoculated with 10% (v/v) pre-culture. The effect of the six different assisted carriers ((2 %( w/v)) on the fermentation was tested for mixed culture. A control batch experiment was done for each support (2 % (w/v)) without culture to measure the adsorption capacity (Q) of each support. The control and main experiment were prepared with the same synthetic medium containing glycerol nearly 25 g/L.
5.3.4 Analytical methods
5.3.4.1 Biomass growth
For the carrier assisted batch dark fermentation the biomass growth was determined using the sum of the cells which are grown freely in the liquid culture and cells attached to the support. A 0.8 mm filter was used to separate the attached cells from the freely suspended ones. After filtering, optical density (OD) at 600nm was measured for the filtrate, which corresponds to the cells which are not attached (free cells).
This residual on the filter was washed with 10 mL deionized water and centrifuged (600 g, 15 min at 4oC) and the supernatant was discarded. Cells attached on support remains as residual on the filter and then re-suspended in 2 mL ultrapure water and dried for SEM and N2 physisorption
analysis. 1mg of this residue (contains attached cells and the support) was used for the scanning electron microscope (SEM) morphological studies. Cell attached on the support carrier was estimated by direct cell counting from the SEM pictures per m2 using ImageJ 1.47i software. The total attached cells were related by multiplying cell counted per m2 of the support by m2 of the support per g.
Cell growth for the free cell experiment and the filtrate from the support assisted (unattached cells) was determined using an optical cell density at 600 nm (OD600). Additionally, cell dry weight
(CDW) was used to quantify the amount of biomass in the serum bottle. CDWs were determined in technical duplicates. 2 x 15 mL culture was sampled and centrifuged (600 g, 15 min at 4oC). Each pellet was re-suspended in 2 mL ultrapure water. CDWs were determined after drying the samples for 1 day in an oven at 105oC. Based on this, OD600 and CDW was correlated using CDW= 857.716
5.3.4.2 Analysis of gas production
During batch experiments the composition of the gas in the serum bottles headspaces were analysed for H2 and CO2 quantification by GC-14B GC, with a thermal conductivity detector (TCD)
and a Carbosive column and a 80/100 Porapak-Q column with Argon (Ar) and Helium (He) as carrier gas with a flow rate of 30 mL/min, respectively. The operational temperatures for the injection port, oven and detector were 150°C, 80°C and 200°C, respectively.
A gastight syringe was used to sample out the gas produced from the processed anaerobic bottle. The syringe has a valve that can be closed to trap the gas within. 0.3 mL volume of gas was taken and equilibrated at an atmospheric pressure and it was calculated against a known calibrated standard of H2 to get the volume gas in percent. In order to ensure the expansion, due to the
pressure inside the bottle a permanent atmospheric pressure was taken as the reference. The H2
gas was identified as a peak with a known pure H2 in the computer software that is connected to
the GC. The peak corresponds to a certain volume percent of H2. The volume fraction of H2 in the
syringe is equivalent to the volume fraction of H2 in the headspace at the time when the gas was
sampled out of the processed serum bottle. The total volume of H2 was calculated multiplying the
volume percentage by headspace of the serum bottle, and thus ideal gas law can be used to calculate the number of moles of gas produced.
5.3.4.3 Liquid metabolites analysis
Fermentation products were identified by HPLC, using Transgenomic column, USA (ICSep ICE- COREGEL 87H3) equipped with Diode array (DAD) and a refractive index (RID) detectors. Aqueous sulfuric acid (H2SO4) adjusted at pH 2.2 was used as mobile phase. Operating conditions for the
HPLC column were 50 °C with a mobile phase flow rate of 0.6 mL/min. Prior to analysis the liquid samples were centrifuged at 9800 rpm for 15 min and filtered through a 0.2 µm disposable filter. The injection volume of the sample was 20 µL. The complete sample elution was achieved within 40 min. Concentrations were determined using standard curves of the respective compounds. Concentrations of product fermentation metabolites were quantified using standard curves of the respective compounds.
GC-MS, was equipped with an HP PLOT column (divinylbenzene/styrene polymer, 30 m long, 0.32 mm ID, 20 µm film thickness) and operating at an inlet temperature of 200°C, a pressure of 6.1 psi and an oven temperature of 35°C for 5 min increasing to 150°C at 5°C/min. Prior the CG-MS analysis the liquid samples were centrifuged at 9800 rpm for 15 min and filtered through a 0.2 µm disposable filter. The injection volume of the sample was 5 µL Glycerol and product fermentation metabolites in the liquid phase were confirmed by GC-MS.
5.3.4.4 Support Characterization
Morphological studies of the mixed culture cells attached on each carrier were examined by scanning electron microscope (SEM, JEOL JSM-840, Japan) at 20 kV and working distance of 15 mm. Prior to SEM observation samples were fixed with 2% w/w glutaraldehyde and dehydrated in graded series of ethanol. Finally, the samples were dried by critical point drier (EMS-850, Japan), coated with gold [31].
N2 physisorption adsorption-desorption isotherms at 77 K was measured using Micromeritics ASAP
2000 equipment for analysis of specific surface area (SBET), average pore volume and pore size.
Prior to the physisorption measurements, all the samples were degassed in vacuum (10 -4 Pa) at 393K. N2 physisorption was used to reveal information about the texture properties for each
carrier before and after the batch fermentation.
X-ray Diffraction (XRD) analysis was used to determine for any change on the surface of the carrier before and after the fermentation. The XRD analysis of the carrier was recorded using a Siemens D5000 diffractometer (Bragg-Bentano for focusing geometry and vertical -goniometer) with an angular 2 diffraction range between 3° and 90°. The samples were dispersed on a Si (510) sample holder. The data were collected with an angular step of 0.03° at 5 s per step and sample rotation. Cu K radiation (=1.54056 Å) was obtained from a copper X-ray tube operated at 40 kV and 30 mA. The crystalline phases were identified using the JCPDS powder diffraction files as a data references.
5.3.4.5 Total organic carbon analysis
The concentration of total organic carbon (TOC) before and after batch fermentation was measured using a Total Organic Carbon Analyzer (Analytik jena, Multi N/C 2100). Prior to analysis the liquid sample was filtered through a 0.2 µm disposable filter.
5.3.5 Data analysis
Data analysis and Kinetic parameters
The adsorption capacity of the adsorbents (support) was calculated based on the concentration change of glycerol in the solution according to Equation (1), where Q is the adsorption capacity (mg/g), Ms is the amount of adsorbent, and QGly,i and QGly,f are the glycerol concentration before
and after fermentation, respectively. Glycerol concentration was determined by HPLC as mentioned above.
Equation (5.1)
A modified Gompertz equation Eq. (2) [32,33] was used to estimate the maximum production rates and the production potentials of the fermentation end products such as: ethanol, acetate, lactate, propionate, succinate, 2, 3 butanediol, CO2 and H2:
,
, , * 1 i max P i max i max i R e P t P exp exp i t P
Equation (5.2)Where Pi(t) is the cumulative production (mmol/L), λ the lag-phase time (h), Pmax,i the production
potential (mmol/L), Rmax,i the maximum production rate (mmol/L*h), t the incubation time (h), and e the exp(1) = 2.718. This equation was found to be suitable for describing the progress of
cumulative production of compounds during the experiments.
Accordingly, for the consumption of glycerol a modified Gompertz equation Eq. (3) [33] was used:
max, 0 max max.
( )
.exp
exp
R
Se
S1
S
S t
S
t
S
Equation (5.3)Where: S0 – initial substrate concentration (mmol/L), S – substrate concentration (mmol/L) at time
t, Smax – maximum concentration of consumed substrate (mmol/l), Rmax,S – maximum rate of
substrate consumption (mmol/L*h). The fitting of the fermentation data was performed using Sigma plot application software version 12.3, where accuracy of the fit was given by correlation coefficients (R2).
For batch cultivation yields of the fermentation end-products, expressed in mole product produced per mole of glycerol consumed, experimental data of substrate adsorbed determined in Equation (1) was considered in the yield calculation.
)
(
max 0 max, max, ads i i PS
S
S
P
Y
Equation (5.4)Where: YPmax,i – substrate yield for fermentation product i, S0 – initial glycerol concentration
(mol/l), Smax – maximum glycerol consumption(mol/L), Sads maximum glycerol adsorbed by the
support (mmol/L)
Maximum specific production or consumption (qmax,i) was calculated using the values obtained from the data fits (Eq. (2) and Eq. (3)), according to Eq. (5) and (6) respectively , and were
calculated using the ratio of the maximum production rate or substrate consumption rate to maximum dry cell weight (DCWmax).
max max, max,
DCW
R
q
i
i Equation (5.5) max, max, max i s S q DCW Equation (5.6)Where: qmax, I specific production or consumption rate i (mmol/l*h), Rmax,i – the maximum
production rate i, maximum substrate consumption rate (mmol/L*h) and DCWmax maximum dry
cell weight (g/L).
Carbon balances (C-balance) and a degree of reduction balances (ε-balance) were calculated
according to Oh et al. and Converti et al. using the elemental biomass composition CH1.74O0.33N0.23
[34,35]. This corresponds to a biomass carbon content of 53.6% and with a degree of reduction of 4.32 electrons per C atom. The reduction degree (ε) was calculated from the following equation (7) [34, 35].
4C
H
2O
3N
Equation (5.7)Where C, H, O, and N denote the atomic coefficient of the chemical formula of a compound. Glycerol conversion (E) by the strains was calculated using the following equation (Eq 8):
100
)
(
0 max 0
S
S
S
S
E
ads Equation (5.8)Determining hydrogen and CO2 production. The number of moles of gas (n) injected into GC at
a room temperature was calculated using ideal gas law in Equation 9.
PV nRT Equation (5.9)
Where, P is atmospheric pressure, V is the volume determined by the injection, R is universal gas constant and T is RT.