Metabolic Profiling of Geobacter sulfurreducens during Industrial Bioprocess Scale-Up

Abstract

During the industrial scale-up of bioprocesses it is important to establish that the biological system has not changed significantly when moving from small laboratory-scale shake flasks or culturing bottles to an industrially relevant production level. Therefore, during upscaling of biomass production for a range of metal transformations, including the production of biogenic magnetite nanoparticles by Geobacter sulfurreducens, from 100-ml bench-scale to 5-liter fermentors, we applied Fourier transform infrared (FTIR) spectroscopy as a metabolic fingerprinting approach followed by the analysis of bacterial cell extracts by gas chromatography-mass spectrometry (GC-MS) for metabolic profiling. FTIR results clearly differentiated between the phenotypic changes associated with different growth phases as well as the two culturing conditions. Furthermore, the clustering patterns displayed by multivariate analysis were in agreement with the turbidimetric measurements, which displayed an extended lag phase for cells grown in a 5-liter bioreactor (24 h) compared to those grown in 100-ml serum bottles (6 h). GC-MS analysis of the cell extracts demonstrated an overall accumulation of fumarate during the lag phase under both culturing conditions, coinciding with the detected concentrations of oxaloacetate, pyruvate, nicotinamide, and glycerol-3-phosphate being at their lowest levels compared to other growth phases. These metabolites were overlaid onto a metabolic network of G. sulfurreducens, and taking into account the levels of these metabolites throughout the fermentation process, the limited availability of oxaloacetate and nicotinamide would seem to be the main metabolic bottleneck resulting from this scale-up process. Additional metabolite-feeding experiments were carried out to validate the above hypothesis. Nicotinamide supplementation (1 mM) did not display any significant effects on the lag phase of G. sulfurreducens cells grown in the 100-ml serum bottles. However, it significantly improved the growth behavior of cells grown in the 5-liter bioreactor by reducing the lag phase from 24 h to 6 h, while providing higher yield than in the 100-ml serum bottles.

FIG 1

Growth curves of G. sulfurreducens grown on NBAF at 30°C for 168 h in 100-ml serum bottles (red line) and in a 5-liter bioreactor with (green line) and without (blue line) nicotinamide supplementation. Relative peak areas (normalized to an internal standard) of nicotinamide in the medium (purple line) detected by GC-MS in the 5-liter bioreactor supplementation experiment are plotted against the sampling time. The time point measurements for the serum bottles are means of three biological replicates with error bars indicating the standard deviation. Single measurements were recorded for the bioreactor samples due to volume constraint.

FIG 2

PC-DFA score plot of the FTIR data collected for all the samples. Five PCs with a total explained variance of 97.39% were used for the DFA. The two sets of samples, i.e., the 100-ml serum bottles (circles) and the bioreactor (crosses), displayed similar growth patterns. However, compared to cells grown in the serum bottles, the bioreactor-grown cells displayed an extended lag phase (up to ∼24 h) and consequently entered the stationary phase at a later time point. The arrows illustrate the main trajectories with respect to time. The color bar on the right represents the duration of the incubation during which samples were collected.

FIG 3

PC-DFA score plot (using 3 PCs with a total explained variance of 96.18%) of G. sulfurreducens cells grown in 100-ml serum bottles (circles) and a 5-liter bioreactor (crosses) between 0 and 48 h of incubation at 30°C. There is a clear separation between the two different sets of samples on the basis of DF1 in the first 24 h of incubation. The color bar on the right represents the duration of the incubation during which samples were collected.

FIG 4

(a) PC-DFA score plot of the G. sulfurreducens cell extracts analyzed using GC-MS from 100-ml serum bottles (stars) and a 5-liter bioreactor (circles), collected from all time points. (b) CCA plot against time of the relative peak areas of detected metabolites by GC-MS for 100-ml serum bottle (stars) and bioreactor (circles) samples. Both samples displayed an overall linear time-dependent correlation. The color bars on the right represent the duration of the incubation during which samples were collected.

FIG 5

CCA loading plot of all the sample extracts analyzed by GC-MS, showing the most significant metabolites contributing toward the positive and negative correlation with time throughout the data set. The list of identified metabolites can be found in Table S2 in the supplemental material. Fumarate is seen as three peaks, as different derivatization products are seen in GC-MS.

FIG 6

Relative GC-MS peak intensities of significant metabolites between 100-ml serum bottle (stars) and bioreactor (circles) cell extracts during the incubation period. The color bars on the right represent the duration of the incubation during which samples were collected.

FIG 7

Pathway of acetate metabolism in G. sulfurreducens during growth on NBAF medium with fumarate as the electron acceptor and acetate as the electron donor. Acetate is transported into the cells (green arrow) via acetate permease (magenta protein channel). Imported acetate can be activated via one of the following two pathways: (i) the acetate kinase (EC 2.7.2.1) followed by the activity of phosphotransacetylase enzyme (EC 2.3.1.8) (purple arrows) producing acetyl-CoA which is directed toward pyruvate synthesis and subsequently into biomass and amino acid synthesis pathways, or (ii) acetate being oxidized through conversion of succinyl-CoA to succinate via the activity of succinyl-CoA:acetate CoA-transferase enzyme (EC 2.8.3.18), resulting in the production of acetyl-CoA (yellow arrows) which is directed toward the TCA cycle. The fumarate provided in the medium is taken up (green arrow) via the fumarate transporter proteins (C₄-dicarboxylic acid transporter). Imported fumarate can either be (i) directed toward the TCA cycle (blue arrow), which operates as an open loop and ends with the formation of succinate and its excretion into the medium (red arrow), or (ii) reduced to succinate via the activity of inner membrane-bound FrdCAB enzyme (fumarate reductase activity) followed by its excretion into the medium (red arrow) (66, 67, 76, 77).

FIG 8

Comparison of the intensities of significant FTIR vibrations identified by the PC-DFA loading plot of serum bottle (stars) and bioreactor (circles) samples during the first 48 h of incubation (see Fig. S2 in the supplemental material). (a) The 1,655 cm⁻¹ amide I region due to stretching of C=O bonds; (b) the 1,402 cm⁻¹ region due to symmetric stretching of C=O bonds in carboxylic acids. Data points represent the mean of the three replicates, with bars indicating the relative standard deviation. The color bars on the right represent the duration of the incubation during which samples were collected.